Improved large-scale immunomagnetic separation device

Abstract

An apparatus and method for immunomagnetic separation having a flexible, expandable processing chamber and a magnetic array.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 930,917, filed on November 5, 2019, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to large-scale cell separation, and more particularly to immunomagnetic separation of cells, and to process innovations for making such separation faster, more efficient, and more economical. [Background technology]

[0003] With the emergence of methodologies and new technologies that allow for the harvesting of mammalian cells and their conversion into biopharmaceuticals capable of eliminating harmful cells in the host, there is renewed interest in the isolation of key initiation cell subsets from peripheral blood mononuclear cells (PBMCs) for these processes. Large-scale clinical cell isolation typically involves batches of 10 9 ~10 11 It is possible to process individual cells. T cells, a key subset for producing genetically modified cells (CAR-T cells) in this new field of cell and gene therapy, typically make up 30-45% of leukocyte transfusions. To obtain enough T cells to begin the production of these biopharmaceutical inoculum, 10 10 A single PBMC (peripheral blood mononuclear cell) is sufficient. On the other hand, isolating stem cells corresponding to 0.5-2% of the starting cell population requires 10 times more initial cells. In either case, immunomagnetic separation requires the following three essential steps: (1) labeling target cells with magnetic material; (2) separating such cells from the mixture; and (3) recovering the target cells. A potential fourth step is possible if separation of the magnetic material from the target cells is desired.

[0004] The jointly owned separation system 1 described in International Publication No. 2016 / 183032 (hereinafter, "'3032"), shown in Figure 1, comprises the following elements: (1) a combination of incubation, separation, and a thin linear fluid chamber (FC) 2 that swirls at an intermediate point to facilitate process steps during purification, such as mixing reagents, performing magnetic separation against gravity, moving buffer and buffer meniscus on the collection surface, and using a beneficial orientation for filling and emptying the chamber; and (2) a magnetic array 5, when in contact with the FC2, particularly suitable for carrying out subsequent purification steps of (a) enhanced loading of magnetic nanoparticles onto target cells, (b) cell separation, and (c) these magnetically immobilized separated cells. As disclosed in '3032, item "(c)" includes a process step of removing bystander cells typically encompassed during magnetic separation, which, unlike other external magnetic field magnetic separations, does not require a suspension and magnetic separation cycle to remove products of such bystander cells. Accompanying bystander cells are instead removed from the magnetically held cells by passing the buffer and the buffer meniscus over it. Thus, the disclosure of '3032 details a method and operation for efficiently performing immunomagnetic cell separation in a minimum number of steps. System 1 of '3032 can perform similar separations for other entities. Note that '3032 discloses System 1 in which the magnetic array 5 and FC2 can be coupled and uncoupled to perform the various steps used in the separation process.

[0005] System 1 described in 3032 is not only useful for large-scale isolation, but can also be used for smaller quantities as it can use FC2 with different collection surface areas, and therefore can be simply scaled up or down. Disclosed System 1 is also designed for use on cells labeled with highly magnetic colloidal nanoparticles that can be separated from blood vessels using an external magnetic gradient field. Cell separation using significantly larger magnetic beads (approximately 1–5 μm) for cell labeling can also be favorably carried out by the method disclosed in 3032, although additional steps are generally required.

[0006] The results of all these considerations are shown in Figure 1, which shows a three-quarters diagram of the main components of the cell separation system 1 disclosed in 3032. Item 2 is a collection chamber having thin, rigid sides with ports 3 for introducing or removing fluid. Chamber 2 is fitted into a frame 4 so that it can be connected to rotary actuator assemblies and linear actuator assembly carriages, as disclosed in 3032. Thus, the plane of the processing chamber 2 can be rotated and translated laterally, as indicated by arrows 9 (lateral movement) and 10 (rotational movement). Also shown in the system 1 is a magnetic array 5 having an iron backplate 6 that provides a yoke and holds fixed magnetic elements 7, which can be arranged in various ways, such as a parallel row of magnetic elements having north and south poles facing the backplate 6. As disclosed in 3032, such an arrangement forms a strong magnetic gradient that attracts the magnetic elements to its plane defined by these pole surfaces. Furthermore, finite element calculation analyses of various magnetic configurations are disclosed, demonstrating that the magnetic gradient can be well controlled with respect to the distance from the plane and the gradient as a function of magnetic reach and retention. The magnetic array 5 can be fixed in space with the magnets 7 facing downward, preferably at an angle of 45°. Alternatively, the magnetic array 5 can be fixed to the processing chamber 2 so that both the magnetic array and the processing chamber can rotate or translate as a unit. Clearly, when the magnetic array 5 is in contact with the processing chamber 2, a strong magnetic gradient is imposed on the contents of the processing chamber.

[0007] Detailed protocols and methodologies for using System 1 to isolate CD3+ T cells from peripheral blood mononuclear cells (PBMCs), as well as the advantages of the rotational and translational properties of the processing chamber 2, were disclosed in '3032'. Briefly, a processing chamber 2, positioned nearly vertically, is filled with a certain volume of PBMCs and a second volume of anti-CD3 FF, which are mixed by rotation and / or translational vibration of the processing chamber 2, during which time the CD3 T cells are magnetically labeled. Next, the processing chamber 2 is translated and brought into contact with the magnetic array 5, bringing the magnetically labeled cells onto the upper inner surface of the processing chamber 2, and the cells are held sufficiently tightly so that the fluid passing over the cells within the processing chamber 2 does not remove the cells. The buffer and meniscus that gently pass over the thus held target cells, with this moderate agitation, can return bystander cells encompassed during the magnetic separation step to the suspension, resulting in a very high level of purity, as disclosed in '3032'. After two or three cycles of such washing (or "meniscus scrub"), the processing chamber 2 can be oriented vertically, the magnetic array 5 can be removed, and the product cells can be suspended in some desired volume of buffer using the rotational and lateral translational properties of system 1. [Overview of the project]

[0008] According to the present invention, the applicant recognized that, due to the need to apply the maximum magnetic gradient to the contents of the processing chamber, the gradient generated by the planar magnetic array decreases sharply as a function of the distance from those planes, thus requiring the collection walls of the processing chamber to be very thin. This thinness not only makes such fragile chambers expensive because they are difficult to manufacture and sterilize, but the thin walls also prevent the processing chamber from becoming distorted during process steps that require the processing chamber to rotate or oscillate from side to side, for example, to perform mixing operations or "meniscus scrubbing" processes (referring to the process of passing a buffer meniscus over magnetically held target cells to achieve high purity). It requires a support system.

[0009] According to the present invention, the applicant also recognizes an imaginative solution to the problems of these chambers by forming a chamber with narrow rectangular rigid walls from a flexible bag, for example, a blood bag of appropriate size. This solution can be achieved by enclosing such a bag between nearly parallel walls and placing the bag under moderate pressure, thereby bringing the walls of the flexible bag into close contact with the enclosing walls and providing a rigid chamber with a wrinkle-free collection surface. The need for a flat, wrinkle-free surface cannot be overemphasized, as the type of magnetic gradient used here decreases sharply as a function of the distance from the plane magnet. Wrinkles on such a collection surface result in a significant loss of target cells, as their height on the plane magnet array is a reduced magnetic gradient. It should be noted that, in addition to using a synchronous pump to maintain the collection processing chamber under pressure and form a rigid chamber, a simple ballast-type auxiliary chamber of appropriate volume, filled with pressurized air and fitted with a pressure limiting valve, can be used. Large (2L) blood bags fitted with appropriate limiting valves have been successfully used.

[0010] As further disclosed below, the applicant has inventively devised a way to couple a magnetic assembly to a processing chamber with narrow rectangular rigid walls, in the form of a bag, such as a blood bag, positioned between substantially parallel walls, and to couple the bag under optionally moderate pressure. The coupling between the magnetic assembly and the processing chamber in the form of a bag may be permanent, with the gradients of the magnetic assembly and the bag perfectly aligned.

[0011] In another embodiment, the present invention can provide a device in which a processing chamber with narrow rectangular rigid walls in the form of a bag, such as a blood bag, arranged between substantially parallel walls, and a magnetic assembly can be optionally coupled and uncoupled with the precise alignment required for reproducible processing. Such a configuration presents significant challenges for the following reasons: (1) the processing chamber is a large, thin-walled, narrow, linear chamber operated under pressure that must maintain its shape throughout the coupling and uncoupling cycle; and (2) the magnetic gradient generated by the magnetic array drops rapidly from its plane, making its proximity to all areas of the collection surface of the processing chamber a significant problem. In one embodiment, disclosed herein is a system that accomplishes the task of coupling and uncoupling these key components with the precision required for reproducible processing. The configuration of the exemplary system disclosed can be easily automated by using commonly available actuators. In addition, gravity can also be used, as the separation system can utilize gravity in the cell separation process through its swirling capability, or to assist in coupling and uncoupling the magnetic array and the processing chamber.

[0012] The reversible coupling between the magnetic assembly and the processing chamber can be achieved by fabricating a novel, independent housing for the processing chamber having a strong cover, preferably transparent, so that the processing chamber can be observed during processing, and a floor or bottom platform having a unique structure that allows close contact between the processing chamber and the magnetic assembly, i.e., the processing chamber and the magnetic array. The bottom floor can be achieved by starting with a rigid rectangular plate, preferably aluminum, that is somewhat larger than the dimensions of the backing plate of the magnetic array, and cutting a rectangular area approximately in the center of the plate, so that the magnets of the magnetic array can be inserted entirely into the space to such an extent that the tops of the individual magnets are level with the top surface of the plate or floor. A support structure is required to hold the bottom pressurized surface of the processing chamber in place. This support structure is required by (1) arranging thin members arranged horizontally and parallel within the notched space, fixed to two of the opposing surfaces formed by the notches in directions corresponding to the directions of the individual magnets of the magnetic array, thus forming a grille-like structure, and (2) between the individual magnets of the magnetic array, This can be achieved by (3) using support members that can be fitted with sufficient tolerances to allow easy entry and exit into the space, spacing the support members so that they can be fitted either between adjacent magnets or between all other magnets when slotted or grille-shaped floors are placed on the magnetic array, and (4) placing thin sheets, e.g., 1 mm rigid Plexiglas® brand acrylic sheets, on the lattice support structure to form a smooth, perfectly flat bottom containment wall for the processing chamber. By appropriately selecting the thickness of the rigid bottom plate or floor and the height of the block magnets used to construct the magnetic array, the planar surface of these arrays can be made within 0.5 to 1.0 mm of the bottom surface of the processing chamber, thereby effectively providing the maximum magnetic gradient to the contents of the chamber.

[0013] As stated above, considering the teachings of this disclosure, it will be clear that many of the advantages described in '3032, particularly with respect to system automation and the elimination of operator tasks, are also produced by the ability to decouple these two main components, namely the processing chamber and the magnetic array. This technique also has another very important advantage, as it eliminates the need for a separate processing station to magnetically label the target cells before they are introduced, as required by the coupled unit. Decouplering the processing chamber and the magnetic array also simplifies product collection by simply discharging the product from the processing chamber.

[0014] In one embodiment, the present invention can provide a system for magnetically separating a target bioentity from a fluid suspension of target and bystander bioentities 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 a processing chamber on its upper surface, the chamber having an opening which can be filled with a cell suspension having a magnetized or magnetizable target bioentity, and the processing chamber is a fluid chamber having a collection surface. In addition, the system may include a magnetic element attached to the platform and movable relative to the platform, which, at a first selected position on the platform, is magnetically coupled to the processing chamber to apply a magnetic field to the collection surface to attract the target bioentity to the collection surface. A chamber control assembly may also be provided connected to the processing chamber and the magnetic element, and the chamber control assembly may be operable to pivot the separation chamber and the magnetic element around an axis in response to rotation of the platform, moving the magnetic element from a first selected position to a second selected position, the second selected position being further from the platform than the first selected position. The magnetic element may be movable in a direction perpendicular to the upper surface.

[0015] Furthermore, the platform may include one or more posts on which a magnetic element is movably mounted, allowing the magnetic element to move from a first selected position to a second selected position on one or more posts, the second selected position being further from the platform than the first selected position. The magnetic element may be configured to move on one or more posts from the first selected position to the second selected position in response to the rotation of the platform. The magnetic element may be configured to move from the first selected position to a second selected position further from the platform than the first selected position in response to the rotation of the platform. The magnetic element may include an array of magnets, the platform may include a cavity extending through the platform, and the magnetic element is dimensioned to fit into the cavity.

[0016] Furthermore, the system may include a plurality of longitudinal nonmagnetic bars arranged parallel to each other in a spaced-out relationship within a cavity, with a plurality of openings provided between each pair of these longitudinal nonmagnetic bars. The magnetic element may include an array of longitudinally extending magnets dimensioned to fit into each of the plurality of openings when the magnetic element is in a first selected position. The platform may also include a nonmagnetic sheet disposed on and in contact with the plurality of longitudinal nonmagnetic bars to provide a flat surface for engaging with the processing chamber. A cover may be disposed on the top surface to define a space between the cover and the top surface for receiving and holding the processing chamber. In addition, a cam is provided that contacts the top surface and the cover, the cam is rotatable to change the distance between the top surface and the cover, and the cover is movable along a direction perpendicular to the top surface, thereby changing the distance between the top surface and the cover.

[0017] In yet another aspect, the present invention can provide a system for magnetically separating a target bioentity from a fluid suspension of the target bioentity and a bystander bioentity within a processing chamber in the form of a bag. The chamber can have an opening that can be filled with a cell suspension having magnetized or magnetizable target bioentities, and the processing chamber is a fluid chamber having a collection surface. The system is a platform having a cavity extending through the platform from an upper surface to an opposing lower surface, and a plurality of longitudinal non-magnetic bars disposed parallel to each other in a spaced relationship within the cavity, wherein a plurality of openings are disposed between each pair of the longitudinal non-magnetic bars; a plurality of magnets disposed within a plurality of openings within the cavity proximate the upper surface, the plurality of magnets being magnetically coupled to the processing chamber to apply a magnetic field to the collection surface to attract the target bioentity to the collection surface; and can include a non-magnetic sheet disposed on the upper surface and contacting the plurality of longitudinal non-magnetic bars to provide a flat surface for receiving and supporting the processing chamber.

Brief Description of the Drawings

[0018] The foregoing summary and the following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the accompanying drawings.

[0019] [Figure 1] FIG. schematically shows a simplified view of certain components of the cell separation system disclosed in ‘3032. [Figure 2] FIG. schematically shows a top view of a standard blood bag modified to optimize its use as a processing chamber for immunomagnetic cell purification in the apparatus of the present invention. [Figure 3A]A side view schematically showing an expandable flexible chamber in the form of a collapsed flexible blood bag, provided with an inflated airbag ready to form a processing chamber by pressurizing the blood bag, disposed, for example, within a "wall-enclosed" frame according to an exemplary configuration of the present invention. [Figure 3B] A side view schematically showing the inflation of the flexible blood bag of FIG. 3A within the "wall-enclosed" frame of FIG. 3A to form a rigid processing chamber. [Figure 4] A top view schematically showing an exemplary configuration of a slotted rigid non-magnetic plate according to the present invention, which can support one side of the pressurized expandable blood bag processing chamber of FIG. 3B. The slots in the non-magnetic plate allow the magnets of the array to come into close contact with the processing chamber. [Figure 5A] A top view schematically showing an exemplary configuration of a planar magnetic array according to the present invention, in which the magnets of the planar magnetic array can be "meshed" with the non-magnetic plate of FIG. 4. [Figure 5B] A side view schematically showing the magnets located on the backing plate of the planar magnet array of FIG. 5A. [Figure 6A] A side view schematically showing the planar magnet array of FIGS. 5A - 5B mechanically connected to the support plate of FIG. 4. A cover plate is added on the support plate to form a frame for holding an expandable flexible chamber such as a flexible blood bag. [Figure 6B] A partially cut-away view schematically showing the system of FIG. 6A rotated 180° about its axis of rotation with respect to the orientation shown in FIG. 6A. This rotation moves the magnets through the gaps in the slotted non-magnetic plate. [Figure 7] An exploded isometric view schematically showing an exemplary configuration of a rotatable magnetic separation device according to the present invention, including the planar magnetic array and the processing chamber of FIGS. 4 - 6B. The swivel / rotation operation serves to engage or disengage the magnets of the array during rotation due to the action of gravity. The part of the rotatable magnetic separation device that houses the expandable flexible separation processing chamber is shown in an exploded manner. [Figure 8] This figure schematically shows a cross-sectional view of the apparatus in Figure 7 when it is not disassembled, cut along cutting line 8-8, when the framework supports the separation chamber while the separation chamber is in close contact with the planar magnetic array. [Figure 9] This diagram schematically shows an exemplary configuration of a cam mechanism according to the present invention, which allows the depth of the separation processing chamber to be changed. [Figure 10] This figure schematically illustrates how the rotation of an exemplary system of the present invention can be used to bring the processing chamber into contact with the magnetic array or to disengage it in order to carry out an immunomagnetic separation procedure step. [Modes for carrying out the invention]

[0020] Referring to the drawings, in which similar elements are numbered similarly throughout, one exemplary method for achieving many of the advantages of both the separation system having the magnetic array 55 according to the present invention and a flexible, inflatable processing chamber 16, for example, a blood bag, is to permanently fix these components to each other (Figures 2, 3A, and 3B). Another exemplary method is to make the processing chamber / bag 16 and the separation system having the magnetic array 55 movable relative to each other (for example, Figures 2, 6A, and 6B).

[0021] Referring first to the chamber 16, Figure 2 shows an exemplary flexible, inflatable processing chamber 16 according to the present invention, which may be provided in the form of a modified blood bag 16 suitable for use in both configurations of Figure 3A and Figure 6A. The modified blood bag 16 may include inlet ports 17 and outlet ports 18 on both sides of the bag (but other positions may be used) for connection to inlet and outlet pumps, respectively, so that a fluid (solution or gas) is pumped into or out of the bag 16 independently or synchronously. It should be noted that the corners 19, 20 of the bag 16 are welded to block the flow to the corners of the bag, as the process may be hindered if there is no good fluid flow. Flow diverters 21 and 22 may be designed to facilitate plug flow through the bag 16. A key advantage of using blood bags as processing chambers 16 in clinical cell separation is that these bags are compatible with blood products, sterile, well-known to users, and inexpensive and disposable.

[0022] Referring to Figure 3A, a system 100 according to the present invention is shown, which includes a flexible, inflatable processing chamber 16, shown as a deflated blood bag, positioned between parallel retaining walls, one of which is shown as 23' and the other as a magnetic array 55. The inflatable processing chamber 16 is pressurized to form a rigid processing chamber, and the walls of this chamber are made flush with the retaining wall 23' and the magnetic array 55 within the frame 23 (Figure 3B). Under moderate hydraulic or pneumatic pressure (<1.0 psi), the blood bag 16 can be kept rigid, and the flexible walls remain open throughout all processing steps. It's wrinkle-free throughout.

[0023] The ballast airbag 25 may be closed and held by a line clamp 26 as one possible method for pressurizing the blood bag 16 (Figure 3A). As described above, one wall of the frame may include a magnetic array 55 of magnets 57, on which a thin (approximately 0.5-1 mm thick) rigid nonmagnetic sheet 52, such as a Plexiglas® brand acrylic sheet, is placed, for example, to create a smooth, flat surface. The opposite side 23' may be easily removable so that a flexible, inflatable processing chamber 16 is inserted and held in place within the frame 23 once pressurized.

[0024] Figure 3B schematically shows the apparatus of Figure 3A, in which the flexible, inflatable processing chamber 16 is inflated within the frame 23. The line clamp 26 is released, and air from the airbag 25 inflates to pressurize the blood bag 16, forming a rigid expansion chamber 16. With the blood bag 16 pressurized, the proper arrangement of the wall 23', the rigid, non-magnetic sheet 52, the magnetic array 55, and the blood bag 16 is achieved so that a magnetic field can be delivered to the processing chamber / bag 16 to carry out, for example, a magnetic cell separation process. Using a synchronous pump connected to a pressure gauge with feedback, the inflatable, flexible chamber 16 can be maintained rigid and under constant pressure for the entire process, typically 50 to 160 minutes, or even longer if additional steps are involved.

[0025] In a separation apparatus 100 constructed by permanently binding a processing chamber 16 to a magnetic array 55, positive cell selection using indirect magnetic labeling can be carried out as follows: (1) a cell suspension can be mixed with a labeled monoclonal antibody (mAb) and incubated in a suitable external system bound to system 100; (2) unbound mAbs can then be removed by centrifugation if necessary; (3) common 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 where separation can be performed immediately; (5) non-target bystander cells entrained during magnetic separation can then be removed from the magnetically collected cells by performing 1-3 cycles of meniscus scrubbing (as disclosed in, for example, 3032) on the separated magnetically held cells; (6) finally the processing chamber is removed from the magnetic gradient and the cells are recovered (in the case of positive selection).

[0026] To make the system 100 shown in Figures 3A and 3B more convenient for magnetic processes such as immunomagnetic separation, it is desirable to have the magnets 57 of the magnetic array 55 in close contact with at least one wall of the modified blood bag 16.

[0027] In this regard, and in another aspect, the present invention can provide a retaining wall for a processing chamber / bag 16 and a support structure 30 that innovatively achieves the need shown in Figure 4. Figure 4 shows a large, rigid rectangular non-magnetic base plate 31, for example, an aluminum plate 7-8 mm thick, having a cavity 32 disposed inside. The dimensions of the cavity 32 may be slightly larger than the dimensions of the processing chamber / bag 16, so that a magnetic gradient is applied to all parts of the bag 16. Multiple non-magnetic bars 33, for example, 1.5 × 12 mm aluminum flat bars, are spanned across the cavity 32. The bars 33 may be held in place within the cavity 32 by slots disposed within the non-magnetic plate 31 surrounding the cavity 32, where each end of the bars 33 is positioned (Figure 4).

[0028] The spacing 34 between adjacent bars 33 is one or more block magnetic fields of the planar magnetic array 55. The holes must be large enough so that the stone 57 can be smoothly inserted into the space (Figures 6A and 6B). Holes 36 may be provided at the four corners of the base plate 31 to secure posts 65 that protrude above the base plate 31 to hold the cover 42 surrounding the processing chamber / bag 16 in place (Figures 4 and 7). Holes 36 may also be used to secure posts 43 that protrude below the base plate 31 at each of the four corners (Figure 7). The posts 43 may be used to suspend the magnetic array 55, as will be described more thoroughly below in relation to Figure 7. In Figures 4 and 5A, the median line 35 indicates the line in which the base plate system 30 rotates to produce the orientation required for the process for cell purification.

[0029] In yet another embodiment, the inventors recognize that permanently coupling the processing chamber 16 with the magnetic array 55 presents some process drawbacks to the system 100 in Figures 3A and 3B. For example, if the processing chamber 16 and the magnetic array 55 can be easily separated, the specific process of bringing the magnet 57 into or out of contact with the chamber 16 can operate as follows: In the uncoupled state between the array 55 and the chamber 16, a cell mixture can be introduced into the processing chamber 16 (and then appropriately enclosed and pressurized with walls), followed by labeling with a monoclonal antibody (mAh). The system 100 may then be oscillated from side to side around a pivot point for mixing and incubation, then a common trapping magnetic material is introduced into the processing chamber 16, which is then oscillated again for mixing and incubation. (Note that this final step cannot be performed while the array 55 and chamber 16 are coupled, because much of the separation of the added magnetic material is likely to occur immediately after the introduction of the magnetic material and before the system is sufficiently mixed. Subsequently, after the introduction, mixing, and incubation of the target cells and magnetic agent, there may be steps in which the processing chamber 16 and magnetic array 55 are coupled and uncoupled. For example, intermittent coupling (20-30 seconds) between the processing chamber 16 and magnetic array 55 can be used to increase the movement of magnetic nanoparticles due to the changing magnetic gradient, while larger cells remain relatively stationary, potentially allowing target cells to find any uncoupled magnetic nanoparticles and increasing the magnetic load on the target cells.)

[0030] In the case of a positive selection, after the used cells have been removed from the processing chamber 16, the magnetically held cells are buffer-washed while the chamber 16 and magnetic array 55 remain engaged, followed by meniscus scrubbing to obtain a highly purified cell product. Bystander cells accompanied by magnetic separation can be easily removed by holding the magnetically separated cells (targeted cells and accompanied cells) in place and passing buffer and buffer meniscus over them, thereby moving the accompanied cells into the fluid phase, thus resulting in highly purified cells that can be easily collected by detaching the processing chamber 16 and magnetic array 55 and draining the chamber 16. In addition to all the aforementioned advantages of a system having a detachable magnetic array 55 and chamber 16, the extra step of removing the processing chamber 16 from its walled-enclosed space, as required in a coupled unit, is eliminated.

[0031] The challenges in fabricating the support structure for the processing chamber wall that contacts the magnetic array 55 are not only because the wall is thin, preferably less than 1.0 mm, but also because the processing chamber 16 is pressurized. 7 1 × 10⁶ cells / mL at an optimized separation concentration. 10 In such a chamber 16, which is confined to a depth of 8-10 mm to accommodate magnetic separation of individual cells, the thin-walled collection surface area is approximately 500 cm². 2 This is possible. At an internal pressure of 0.8 PSI, a load of approximately 65 pounds is applied to its surface. Maintaining the pressurized collection chamber wall in place and preventing the wall from deforming when the magnetic array 55 is not pressing against it is important for coupling and connecting the array 55 and the chamber 16. This can lead to uneven collection of target cells, as well as the strain that may occur on its surface during uncoupling. Preventing uneven collection of target cells and / or strain on the chamber wall presents a significant challenge. In addition, a means for coupling and uncoupling the array 55 and chamber 16 in a relatively simple manner that can be automated would be most advantageous.

[0032] It is important to note that the surface area calculated above for the collected surface is in good agreement with another important consideration of the system of the present invention. A considerable amount of experimental data has been accumulated showing that bystander cells encompassed during magnetic separation can be efficiently removed by meniscus scrubbing only when the number of collected cell layers is about 6, possibly less than 7. In this case, 10 of the layers 10 To isolate 40-50% of the total cells of an individual, approximately 520 cm² is needed to meet the required conditions. 2 It is easy to demonstrate through further consideration that a certain collection area is necessary. Therefore, the collection surface needs to be large enough to accommodate the collection of the desired cells, which should be less than 7 cell layers.

[0033] Depending on the advantages recognized above for a system in which the array 55 and the chamber 16 can be coupled and uncoupled, in another embodiment, the present invention provides an apparatus capable of reversibly coupling a flexible, expandable processing chamber 16 with the magnetic array 55. For example, in one exemplary configuration, the apparatus of the present invention may include suspending the magnetic array 55 below the base plate 31 via cylindrical posts 43, along with corresponding sleeve bearings 44 mounted at the four corners of the base plate 31 (Figures 6A to 8). The length of the posts 43 should be long enough so that no magnetic gradient is imposed on the processing chamber / bag 16 when the magnetic array 55 is in its lowest position (Figures 6A, 7).

[0034] That is, when the magnetic array 55 is suspended below the base plate 31 on the post 43 and sleeve bearing 44, gravity causes the magnetic array 55 to be in its lowest position on the cylindrical post, i.e., completely disengaged from the processing chamber / bag 16 (Figures 6A and 7). On the other hand, when the apparatus 40 is inverted, gravity causes the magnetic array 55 to slide down into the slotted grid of the housing floor and thus engage. Thus, one embodiment of coupling and disengaging described in this way may be gravity-driven, thereby allowing the system to accomplish its task and do so favorably for magnetic separation protocols, such as those disclosed in 3032.

[0035] If system 40 is mounted on a pivot bar fixed to the magnetic array 55, it will be clear that engagement of the magnetic gradient occurs in the orientation opposite to the orientation described above. Gravity can be helpful for all needs, but if fully automated engagement / disengagement is desired in any orientation, a simple actuator can be used. Gravity assistance can also be part of the automated system, as it requires a less energy actuator. It may also be advantageous to provide a locking position in the system described in this way. For example, if the magnetic array 55 is on top by rotating the described system by 180°, a locking mechanism can be used to hold the two components in a fixed position, keeping these components engaged regardless of how the system is oriented.

[0036] By having a pressurized collection / processing system and a system that allows coupling or uncoupling of a magnetic gradient, all embodiments disclosed in '3032' can be easily achieved using the following steps: (1) Using an empty pressurized processing chamber, reagents and cells are pumped into the system at a suitable angle (around 45°) in uncoupling mode; (2) Reagents are mixed and agitated with target cells by swirling the processing chamber in the absence of a magnetic field; (3) Magnetic separation of the target can be made to occur against gravity over a wide range of beneficial angles that hold the units together; (4) Processing steps such as meniscus scrubbing of magnetic cells are performed with the magnet engaged and the coupling gravity driven. In some cases, the magnets can be rotated through the angle at which they are engaged to (5) disengage the unit so that the product can be collected. Systems having the ability to arbitrarily position the contents of the processing chamber in a gradient magnetic field also provide another option of the system, namely, the option of suspending the magnetically collected cells in the presence of a washing buffer, where the magnets can be disengaged, rotated through their corresponding angles, and then the processing chamber can be reoriented to carry out a second or third suspension and magnetic separation. Thus, step 4 provides another method for removing encompassed bystander cells.

[0037] In addition, means for changing the depth of the processing chamber are disclosed, which can be used to reduce the processing reagent after magnetic separation has been performed. This possibility allows many reagents and time-saving methods to be incorporated into the separation scheme. For example, in the case of complete apheresis product separation, the typical volume for separation in this system is about 330 mL, and the separation can be performed in a processing chamber with a depth of 7 mm. In positive separation, magnetically collected cells must be immersed in a clean buffer and then undergo at least two cycles of meniscus scrubbing requiring at least 660 mL of buffer. By lowering the depth of the processing chamber after magnetic separation to about 3 mm and reducing the required volume of buffer by more than half before adding the wash buffer, the time for adding and removing buffer from the processing chamber is reduced accordingly, and all of these times are important from an economic standpoint as well as from a cell viability standpoint, as reduced processing time means the most reliably viable cells.

[0038] The terms flexible bag, collection or processing chamber, and blood bag with port are used interchangeably. The external magnetic gradient is the magnetic gradient formed in free space using magnetic pole pieces, their polarity, their specific arrangement, and power, all of which can be used to generate a spectrum of very different gradient magnetic fields in space. 3032 discloses in great detail the analysis of the planar magnetic arrays used herein.

[0039] Exemplary Uses Many magnetic nanoparticles exist that can be used for immunomagnetic separation using the apparatus of the present invention. However, highly magnetic colloidal nanoparticles (HMNPs) in the 140 nm size range, such as those by Liberti et al. (U.S. Patent No. 5,698,271; U.S. Patent No. 6,120,856), which are colloidal and highly magnetic (approximately 84% magnetic mass), are desirable because they can magnetically label cells by diffusion force, and cells thus labeled with HMNPs can be separated in an external magnetic apparatus having a gradient slightly exceeding 4–6 kilogauss / cm. Magnetic nanoparticles of this size (150 nm) are advantageous because the magnetic collection of these thus labeled materials or entities can be well controlled and, in fact, can be collected in a single layer with a uniform magnetic gradient that is readily generated in a radial gradient quadrupole magnetic apparatus, as the inventors have discovered.

[0040] The inventors have further discovered that when target cells are magnetically collected in a monolayer or nearly monolayer, it is not necessary to perform such suspension and magnetic collection cycles of the target to remove bystander accompanying cells, as is commonly done in the art. Instead, target cells collected in a moderately homogeneous layer can be removed by simply passing a buffer meniscus over these cells, which are then magnetically held in place. The inventors refer to this process for purifying the isolated cells as “meniscus scrubbing.” Thus, in a simple secondary purification process, it seems reasonable to suggest that surface tension can act to remove possibly weakly held accompanied non-target cells. Evidence supporting the idea that “meniscus scrubbing” is a very gentle process is found in the inventors' findings that this process does not negatively affect cell viability. Naturally, this results in a higher target cell yield.

[0041] Based on these fundamental discoveries and others, multiple design principles can be incorporated into the apparatus of the present invention: (1) The distance required for target cells to move to the collection wall is desirably made as small as possible so that bystander cell entrainment is minimized (based on experimental data showing that the greater the distance, the greater the bystander entrainment), (2) Principle "(1)" in combination with the need to accommodate the separation of a large number of cells (10 9 ~10 11 cells), requires that cells be collected over a surface area sufficient to layer the cells relatively uniformly in 6 to 7 single layers within a chamber having a small depth (less than 15 mm, within the magnetic gradient), (3) Any pile-up of collected target cells is eliminated or minimized, such that the advantage of collecting cells in a layer where "meniscus scrubbing" can be used essentially indicates that a very large flat plane is used for magnetic collection to accommodate the processable volume required by the cylindrical surface (quadrupole separator).

[0042] To collect target cells in layers, the development of a planar magnetic gradient is required that allows for uniformity over a large surface area (large enough to layer up to 7 single layers of cells). The inventors have shown that by forming such a gradient, target cells can be collected in approximately a single layer. A thin, linear collection processing chamber is formed to conveniently pass a buffer meniscus over a large area of magnetically held cells, and the chamber pivots at its midpoint such that the oscillated fluid and air bubbles therein can flow over such cells and literally scrub off bystander cells. Additionally, the pivoting ability of the chamber is used to perform the various steps of immunomagnetic separation in an optimal manner. For example, reagents can be added to the processing chamber and mixed by rocking left and right, the processing chamber can be tilted to an optimal angle to fill or empty it, and magnetic separation can be performed against gravity, which the inventors have demonstrated leads to a reduction in entrained bystander cells.

[0043] Referring to Figures 5A and 5B, Figures 5A and 5B schematically show block magnets 57 optimally spaced on a magnetically conductive backing plate 56 to generate a strong and nearly uniform gradient force across the area defined by the block magnets 57 and the plane defined by the top of the block magnets 57. Note that this area is equivalent to (slightly smaller than) the cavity 32 of the baseplate system 30. Small holes 38 drilled in the magnetic array 55 hold the post 59 (Figure 9), which securely holds the processing chamber / bag 16 when obtained from a blood bag that has a hole or slit at the top for the bag to be suspended, as many blood bags do. Drilled holes 39 at the four corners of the magnetic array 55 house bearings (not shown) that will allow the magnetic array 55 to be adjacent to the baseplate system 30, as will be described later. Line 37 represents the centerline of the magnetic array 55. Figure 5B shows a side view of the magnet array 55, illustrating how the magnets 57 are spaced apart on the magnetoconductive backing plate 56.

[0044] Figure 6A schematically illustrates an exemplary configuration of a functional, automateable separation system 40, showing in cross-section how the planar magnetic array 55 is bonded to the underside of the base plate 31 via posts 43 and sleeve bearings 44, allowing the magnetic array 55 to slide upward so that pairs of magnets 57 of the magnetic array 55 can pass through the open space 34 of the base plate 31. The processing chamber / bag 16 is shown under pressure. For the sake of simplicity in the drawings of Figures 6A and 6B, thin (0.5-1.0 mm) rigid plastic sheets located below the processing chamber / bag 16 and above the bars 33 are not shown. (The rigid plastic sheets are shown and described in later figures, e.g., sheet 52 in Figure 7.) The inventors have demonstrated that such a rigid sheet 52 in Figure 7, positioned on these support bars 33, processes a perfectly flat surface without distortion even under high pressures of about 4 psi. The chamber / bag 16 is shown to be provided. In Figure 6A, a thick sheet 42 of Plexiglas® brand acrylic sheet, which optionally allows visualization of the contents of the processing chamber / bag 16, provides an upper wall for enclosing the processing chamber / bag 16 during processing. The sheet 42 may be held in place by a wing nut 46 and attached to a post 65 fixed to a drilled hole 36 in Figure 3. The cover sheet 42 can be spring-biased by placing a spring 51 between the wing nut 46 and the sheet 42.

[0045] In Figure 6A, the separation system 40 is shown vertically with the magnetic array 55 suspended from the base plate 31. Note that in this case, gravity positions the magnetic array 55 at its lowest point, i.e., the sleeve 44 rests on the stop 45 on the post 43. When the separation system 40 is swung 180 degrees around the pivot point 41, the system 40 looks as shown in Figure 6B. Figure 6B shows how gravity causes the magnetic array 55 to slide downward so that the magnets 57 move within the space 34 of the plate 31. By appropriately selecting the height of the magnets 57, the thickness of the base plate 31, and the placement of the sleeve bearings 44, the magnets 57 can be positioned at the same height as the surface of the base plate 31 closest to the processing chamber / bag 16. Thus, by simply swung the separation system 40, the inventors have fabricated a system for engaging and disengaging the magnetic array 55 and the processing chamber / bag 16, two main components of the system. Furthermore, in the orientation shown in Figure 6A, it will also be apparent that the magnetic array 55 and the processing chamber / bag 16 may remain disengaged when tilted approximately 90° in either direction. This orientation allows the separation system 40 to be used for mixing reagents in the absence of a magnetic gradient. Similarly, in the orientation shown in Figure 6B, where the magnetic array 55 and the processing chamber / bag 16 are in contact, there is also a wide range of angles over which gravity keeps them connected. This capability allows the separation system 40 to be used for processes such as meniscus scrubbing, as disclosed in, for example, 3032.

[0046] A locking mechanism can be added to hold the magnetic array 55 and the processing chamber / bag 16 together or separately, expanding the utility of the separation system 40. For example, if the separation system 40 is used as shown to perform magnetic separation, the system 40 needs to be inverted as shown in Figure 6B to engage with the processing chamber / bag 16 and the magnetic gradient. Even if separation by gravity has been shown to be beneficial, there may be cases where it is not beneficial or advantageous. By having a locking mechanism to counteract the effects of gravity, the separation system 40 can be locked in the orientation of Figure 6A, rotated 180°, and perform separation by gravity or some other action. Another advantage of the locking mechanism is that it controls how the units come together or separate. For example, without a locking mechanism starting from the orientation of Figure 6A, if the units are rotated and reach an orientation where gravity causes motion, torsional forces may be generated in the post and sleeve bearings, potentially causing wear on these components. A more sophisticated technique is to rotate the device 180° while locked, and then unlock it. This method minimizes wear on components and precisely controls the time the magnetic field is applied or removed.

[0047] Figure 7 shows a depiction 50 of the separation system 40 mounted on a rotatable shaft 54 ​​and positioned within a support structure 53 that allows for 360-degree rotation. This depiction is an exploded view of the components of the system 50 that form the space in which the flexible container, which will become the rigid processing chamber 16, is placed. The grille-like structure of the support bars 33 that support the processing chamber / bag 16 when it is pressurized is a prominent feature of the base plate 31, and the individual support bars 33 and the open spaces 34 between them are clearly shown. Preferably a thin, non-magnetic rigid sheet 52 having a thickness of less than 1.5 mm is placed on top of the bars 33 of the base plate 31 to provide a smooth, flat surface for the sides of the processing chamber / bag 16 that will be pressed against when under pressure. Compression springs 51 are fixed to the base plate 31 by four support posts The spring 51 can be positioned on top of the cover 42 on the corner post 65. The spring 51 is fitted to resist the upward pressure on the cover 42 when the processing chamber / bag 16 is pressurized, and the force must be somewhat large. To fix the depth of the flexible, inflatable processing chamber 16, a set of thick-walled cylinders (not shown) can be positioned on the corner post 65, which can be 3 to 12 mm high for optimal isolation, setting a limit on how close the lower surface of the cover 42 can get to the upper surface of the corner post 65.

[0048] Figure 8 shows a cross-sectional view of the system 50 in Figure 7, cut along the cutting line 8-8. The way in which the magnets 57 mesh in pairs with the support bar 33 and are at the same height as the top of the support bar 33 demonstrates that this exemplary design allows the gradient magnetic field of the planar magnetic array 55 to exert its force on the processing chamber 16 without being suppressed.

[0049] There is one other practical application that can be achieved from the designs and concepts disclosed herein. It may be desirable to be able to vary the depth of the collection chamber during the operations required for the separation protocols described. To illustrate this advantage, a typical complete apheresis product (approximately 7 × 10⁻¹⁶) on this system is shown. 9 The total number of nucleated cells (TNCs) is 350 mL in final volume (2 × 10 at the time of separation). 7 Consider that the cells will be separated into individual cells / mL. For this separation, use 440 cm³. 2 A blood bag with a properly modified surface area is used, and its depth is fixed at 8 mm. The maximum filling or emptying rate of this system is 60 mL / min, because cells are removed by shear force at a higher rate. This takes 6 minutes. Furthermore, after the oscillating meniscus scrub procedure, a 6-8 minute pause is used to recollect any target cells removed during the process. Thus, three meniscus scrub cycles require 6 filling / empty cycles plus 2 pause periods, totaling 60 minutes. In addition, during these cycles, 3 × 350 mL of buffer waste is generated, which becomes part of the hazardous waste.

[0050] On the other hand, if the depth of the processing chamber is set to 8 mm for the initial separation (to accommodate the total number of cells to be processed and to achieve the optimal cell concentration in the separation), and then reduced to a depth of 3 or 4 mm for subsequent processing steps, this not only reduces the total volume of the chamber, but the inventors have also found that in a simulated system, a smaller depth actually provides a more effective meniscus scrub for these subsequent purification steps.

[0051] Therefore, with the depth change described above, the volume is reduced to 131 mL with each fill / empty cycle, thus reducing the time requirements for these steps by more than half. Furthermore, since the removed cells are closer to the collection surface with a gradient nearly twice as large at 3 mm compared to 8 mm, the "pause" period for re-collection is also shortened. It is estimated that the 56 minutes of the process steps described above can be reduced to 20 minutes, which is significant in terms of throughput.

[0052] Figure 9 shows a mechanism for changing the depth of the processing chamber using an elliptical cam 60 attached to a lever 61. By selecting the dimensions of this ellipse, for example 8 × 4 mm, the lever 61 can be moved from an orientation of 8 mm to a dimension of 4 mm, or in any desired ratio. Four such cams 60 and levers 61 may be positioned at each of the corners of the chamber housing unit. Figure 9 also shows a post 59 extending through a magnetic array 55 and a base plate 31 to hold the processing chamber / bag 16 in place within the separation system 40 when the blood bag is obtained from a blood bag that has a hole or slit at the top for suspension, as is the case with many blood bags.

[0053] There are several methods to reduce the depth of the base plate 31 and the inserted inflated blood bag 16 as described above. First, a flexible bag such as the blood bag 16 is placed in system 40 Considering that this bag is under a pressure of approximately 0.7 psi when inserted into the cross section, the cover 42 can exert a total force of approximately 60 pounds, as described above for the larger bag. This force needs to be counteracted by a compression spring 51 that holds the cover 42 in place. A fairly strong spring is needed to drain the fluid from the collection chamber. A better approach would be to adjust the cam 60 to its smaller dimensions and use an outflow pump to perform most of the emptying of the processing chamber, thereby reducing the depth. Nevertheless, with this simple mechanism, the filling and emptying cycles for these processes can be reduced as well as the time required for target cells, which may have been forced into a suspended state during the meniscus scrubbing process, to return to the collection surface.

[0054] Figure 10 shows a schema for performing immunomagnetic separation on a system 50 that uses gravity to engage or disengage a chamber complex 62, which includes an inflatable processing chamber 16, a thick sheet of Plexiglas® brand acrylic 42, and a thin, rigid, non-magnetic sheet 52, and a magnetic array 55 for different steps of the protocol. In Figure 10, the first parallel rectangular panel shows the chamber complex 62 and the planar magnetic array 55 separated so that the magnetic array 55 is held in place by gravity in its lower configuration. In this configuration with the units separated at approximately -45°, the apheresis product, mAb, and magnetic fluid are added sequentially, and the system is oscillated between - / +45° to mix and incubate (top center panel). In the top right panel, the system is rotated so that the magnetic array 55 is on top, here mated with the chamber complex 62, and the separation of magnetically labeled entities against gravity (preferred) takes place. In this same orientation, the product can be recovered (negative selection), or the spent PBMCs can be pumped in and discarded. In addition, in this same configuration, the magnetic array 55 remains mated to the chamber complex 62, and the buffer passing over the magnetically held target cells allows for meniscus scrubbing as the buffer and meniscus pass over them, as shown in the lower left panel, so that the buffer can be added to the collection chamber and the system can be agitated. In this way, bystander-accompanied cells are returned to a suspension state very effectively. After several scrubbing cycles, the desired amount of buffer can be added, the orientation can be reversed with the magnetic array 55 facing downwards (lower right panel), and the cells can be suspended and collected as shown in the last panel. The following examples demonstrate the usefulness of the foregoing disclosure and illustrate innovations that may be used to prepare tumor cell-free T cells or subsets thereof.

[0055] (Example 1) Negative selection of CD3+ cells using magnetic gradient and gravity-driven engagement / disengagement of processing chamber. The frozen apheresis product was thawed at room temperature (RT), centrifuged, and the pellet was suspended in RPMI culture medium containing 10% fetal bovine serum. The suspended cells were centrifuged and suspended two more times in the same buffer, and finally, after a fourth centrifugation in cell separation buffer, 1 × 10⁶ cells were obtained. 8 The cells were suspended to a cell count of 10 cells / mL. 18 mL of this suspension was pumped into a processing chamber / bag 16 (Figure 2) approximately 3 × 6.75 inches, having an inlet port 17 and an outlet port 18 at both ends. The bag (processing chamber) was positioned within a frame 23 between the wall 23' and the magnetic array 55, as shown in Figure 3A and represented in the upper left panel of Figure 10. The depth of the flexible, inflatable processing chamber 16 was set to 8 mm, and the bag was pressurized with compressed filtered air at 0.5 psi before pumping the cell mixture into the bottom port. The upper port 18 was attached to a ballast air bag 25, which was similarly pressurized and had a relief valve set to 0.5 psi. Thus, once the cell mixture entered the bag, the air was released from the system. Next, 18 mL of a proprietary cocktail of mouse monoclonal cells, all IgG1 class and targeting all but CD3+ cells, was pumped into the processing chamber and oscillated as shown in the second panel of Figure 10 to mix the reagents. 2 cycles of oscillation (5 oscillations each) After using the (dynamic) method, a 14-minute static incubation was performed. At this point, 36 mL of 20 μg / mL rat anti-mouse IgG1 magnetic fluid was pumped into the processing chamber, and the processing chamber was agitated as described above. After a 10-minute incubation, 18 mL of separation buffer was added to the processing chamber so that it held 90 mL of suspension. The contents were mixed by agitating the processing chamber as described above, and the system was rotated 180° to engage the magnetic array 55 with the chamber complex 62 (upper right panel of Figure 10), thereby giving the magnetically labeled cells in the processing chamber a strong upward attractive force towards the top surface of the processing chamber.

[0056] After 15 minutes, the non-magnetic fraction (negatively selected cells, i.e., fraction I) was collected and analyzed by flow cytometry. Next, with the magnetic array 55 still above and engaged with the chamber complex 62, the processing chamber was filled with 80 mL of cell buffer, and the system was rotated 180°, thereby disengaging the magnetic array 55 from the processing chamber / bag 16, and the system was oscillated to allow the buffer and large bubbles to pass over the cells and transfer the collected non-target cells to the suspension (Figure 10, lower right panel). This required three cycles of oscillating, each cycle having five oscillations. The system was rotated again so that the magnetic array 55 was above and engaged with the processing chamber 16 to produce a second separation, after which the non-magnetic cells (fraction II) were collected. The analysis of these negative fractions is shown in the table below. The original product was 60.13% CD3+ by flow analysis. [Table 1]

[0057] In this process, it is noteworthy that the apheresis product does not need to be removed before the addition of rat anti-mouse IgG1 FF, a common capture magnetic fluid, and is incubated with a cocktail of mAh and unbound antibodies after incubation. This is a very important advantage, perhaps unique to nanoparticles in this size range, as unbound mAh are typically removed before the introduction of a common capture material. This is likely a result of the high binding ability of these FFs and their size. The inventors have found that these colloidal nanoparticles in the range (135–150 nm) used for magnetic cell separation aggregate very slowly when some agglutinin is added, compared to micron-sized particles.

[0058] (Example 2) Negative selection of CD3+ cells from apheresis products containing circulating tumor cells [CTCs]. Currently, CAR-T cell therapy is the most successful treatment for B-cell cancer. Clearly, apheresis products from such patients are very likely to contain cancerous B cells. However, in negative selection against CD3+ cells, such tumor cells are eliminated along with normal B cells targeted by specific mAh in the appropriate cocktail. Currently, extraordinary efforts are being made in using CAR-T technology for solid tumors. When preparing CD3+ preparations for producing CAR-T cells for such patients, it will be important to ensure that no tumor cells contaminating the negative fraction remain. Solid tumors are Because they are of epithelial origin and have extensive experience in the isolation and identification of circulating tumor cells [CTCs] (Terstappen et al., U.S. Patent No. 7,332,288 B2 and U.S. Patent No. 6,645,731 B2), the inventors hypothesized that adding an anti-epithelial antibody to the incubation mAh cocktail might be advantageous. In this example, an anti-epithelial mAh (clone VU1D9) was added to the incubation cocktail.

[0059] To evaluate the ability of the present invention's system to not only purge CTCs but also remove non-CD3+ cells, in negative selection of CD3+ cells, colon cancer cell line (Colo 205) was spiked into the apheresis product prepared as described above. These cells were fluorescently stained with CellTracker® Red CMTPX Dye (Thermo-Fisher). Based on the fact that metastatic cancer patients can typically have at least 200 CTC / mL of blood, 200 Colo 205 cells were selected, resulting in 3 × 10⁶ cells in the initiation product. 6 Each whole-nucleus cell was spiked. IgG1-class anti-epithelial cell mAh (clone VU1D9) was added to an mAh-dedicated mAh cocktail optimized to deplete all non-CD3-negative cells, in a 0.5 μg / mL cocktail / cell incubation suspension. Separation was performed as described above. The results (yield / purity) for CD3+ cells in the supernatant were almost identical to the data above.

[0060] To test the effectiveness of removing epithelial cells during negative selection, a CTC detection test was performed on 3 × 10⁶ CD3+ cells recovered during negative selection. 6 The procedure was performed on double 5 mL aliquots at a rate of 100,000 cells / mL. If Colo 205 cells were not removed, up to 10,000 cells [(200 / 3 × 10 6 ) × (3 × 10 7 It should be noted that 10,000 tumor cells (x5) will be detected. However, in reality, only 50-60% can be expected. To two 5 mL samples, 8 μg / mL of the magnetic fluid, VU1D9 anti-Epcam, and biotin BSA that were bound to it were added, mixed, incubated for 20 minutes, then 0.8 μg / mL of streptavidin was added, mixed, and incubated for 5 minutes. (The purpose of the last step is for streptavidin to bind unbound FF to FF bound to Colo 205 cells, thereby increasing their magnetic load and significantly increasing their ability to be magnetically separated.) After separation in a quadrupole magnetic separator, the supernatant was discarded, the separation tubes were removed from the magnetic apparatus, and the walls of these tubes were carefully washed with 2.0 mL of buffer to transfer the cells collected for subsequent separation from the sides of the tubes to a volume of 2 mL. This process of reducing the volume while retaining the magnetically collected cells was repeated until the sample volume was 200 μL. Next, the sample was plated onto polylysine-coated glass slides, and cells were counted using a fluorescence microscope. In the control spike experiment, 55% of the spiked cells were captured. No fluorescent cells were detected in the negative CD3+ cell experiment. Considering that the above protocol for CTC detection can only detect about 5 cells / mL of the sample, the addition of anti-Epcam monoclonal to the mAh cocktail of the present invention is clearly an effective means of removing such cells from these preparations. This application may be very important when generating CAR-T cells, as there is a high probability that initiating cells will proliferate and thus have the potential to proliferate CTCs.

[0061] The aforementioned disclosures demonstrate how gravity and compression spring forces can be used to simplify the automation of processes that require many steps. The mating of the cell collection chamber 16 with the planar magnetic array 55 can be performed by various mechanical / electronic elements that require considerable engineering effort and fairly complex manufacturing. The concept disclosed herein eliminates that need. This embodiment not only demonstrates the usefulness of the concept of this apparatus but also demonstrates their application to the preparation of initiating materials for CAR-T cells and other cell applications requiring high purity.

[0062] In addition to using the apparatus of the present invention for immunomagnetic cell separation, a separation / processing chamber The ability of such a device, used to intermittently impart a magnetic gradient to the contents of a cell therapy construct, may be advantageous for another important need in the manufacture of cell therapy constructs. For example, in concurrently pending international patent application 2018 / 022694A1, it was demonstrated that positively selected T cells magnetically labeled with a multivalent common capture agent such as streptavidin magnetic fluid nanoparticles (Liberti et al., U.S. Patent No. 5,698,271, U.S. Patent No. 6,120,856) can subsequently be activated and proliferated by the simple addition of a biotinylated anti-CD28 antibody. In the case of positively selected CD4+ cells in which such nanoparticles are linked to a CD4 epitope via a specific antibody, activation / proliferation requires the addition of two antibodies, namely biotinylated anti-CD3 and biotinylated anti-CD28. It has been demonstrated that the application of an intermittent magnetic gradient in the step in which these latter antibodies are added to the common capture isolated cells results in significantly greater proliferation. Therefore, the device of the present invention may be ideal for its application. Purified, positively isolated cells are suspended in a collection chamber in the absence of a magnetic gradient, an activator is added, and the contents are mixed by oscillating and intermittent magnetic gradients applied, either by simply coupling the collection chamber and the magnetic array via an actuator, or by orientation using gravity to induce coupling or uncoupling.

[0063] Conclusion: The above specific descriptions are intended to illustrate and illustrate the present invention and should not be considered to limit the scope of the invention, which is defined by the literal and equivalent scope of the appended claims.

[0064] Numerous patent and non-patent publications and patent applications are referenced in the aforementioned specification, and the entire disclosures of each of these publications / applications are incorporated herein by reference.

[0065] While specific embodiments of the present invention have been described and / or illustrated above, various other embodiments will be apparent to those skilled in the art from the aforementioned specification. Therefore, the present invention is not limited to the specific embodiments described and / or illustrated, and is subject to considerable modification and alteration without departing from the scope of the appended claims.

Claims

1. A system for magnetically separating a target bioentity from a fluid suspension of target and bystander bioentities in a processing chamber, A platform having a cavity extending through the platform from an upper surface to an opposing lower surface, configured to receive a processing chamber at the upper surface of the platform, the processing chamber having an opening into which the processing chamber can be filled with a cell suspension having a magnetized or magnetizable target or bystander bioentity, and the processing chamber being a fluid chamber having a collection surface, A magnetic element mounted within the cavity of the platform and movable within the cavity in a direction perpendicular to the upper surface of the platform, wherein the magnetic element is movable between a position adjacent to the upper surface of the platform, where it is magnetically coupled to the processing chamber and applies a magnetic field to the collection surface to attract a magnetized target or bystander bioentity to the collection surface, and a position away from the upper surface of the platform, where it is not magnetically coupled to the processing chamber. A chamber control assembly connected to the processing chamber, the platform, and the magnetic element, which is operable to rotate the processing chamber, the platform, and the magnetic element as a single unit 360 degrees around an axis, so that the processing chamber is positioned on or below the platform, A system that includes these features.

2. The platform includes one or more posts, and the magnetic elements are movably mounted on the one or more posts so that they can move toward or away from the upper surface of the platform. The system according to claim 1.

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

4. The system according to any one of claims 1 to 3, wherein the magnetic element is configured to move in response to rotation of the processing chamber, the platform, and the magnetic element as a single unit, and about the axis of the magnetic element.

5. The magnetic element includes an array of magnets. The system according to any one of claims 1 to 4.

6. The magnetic element is sized to fit within the cavity. The system according to any one of claims 1 to 5.

7. The cavity comprises a plurality of longitudinally oriented nonmagnetic bars arranged parallel to each other and spaced apart. The system according to any one of claims 1 to 6.

8. A plurality of openings are provided between each pair of the longitudinal nonmagnetic bars, The magnetic element includes an array of longitudinally extending magnets, which are dimensioned to fit into each of the multiple openings when the magnetic element is in the position adjacent to the upper surface of the platform. The system according to claim 7.

9. The platform includes a non-magnetic sheet disposed on the plurality of longitudinal non-magnetic bars and in contact with the plurality of longitudinal non-magnetic bars, in order to provide a flat surface for engaging with the processing chamber. The system according to claim 7 or 8.

10. A cover comprising a cover disposed on the upper surface such that a space for receiving and holding the processing chamber is defined between the cover and the upper surface, The system according to any one of claims 1 to 9.

11. A cam that contacts the upper surface and the cover, and is rotatable to change the distance between the upper surface and the cover, is provided. The system according to claim 10.

12. A system for magnetically separating a target bioentity from a fluid suspension of target and bystander bioentities in a processing chamber, wherein the processing chamber is a fluid chamber having an opening into which the processing chamber can be filled with a cell suspension having a magnetized or magnetizable target bioentity or bystander bioentity, and the processing chamber has a collection surface, A platform having a cavity extending through the platform from an upper surface to an opposing lower surface, A plurality of magnets disposed within the cavity and movable within the cavity relative to the upper surface of the platform, wherein the plurality of magnets are movable to a position close to the upper surface, where they are magnetically coupled to the processing chamber and apply a magnetic field to the collection surface to attract magnetized targets or bystander bioentities to the collection surface, and to a position away from the upper surface of the platform so as not to be magnetically coupled to the processing chamber, The processing chamber, the platform, and the multiple magnets are connected to each other, and the processing chamber is positioned above or below the platform, with the processing chamber, the platform, and the multiple magnets as a single unit, with an axis A chamber control assembly that can be operated to rotate 360 ​​degrees around the center, Equipped with, The platform includes one or more posts, and the plurality of magnets are movably mounted on the one or more posts so that they can move toward or away from the upper surface of the platform. system.

13. A plurality of longitudinal nonmagnetic bars arranged parallel to each other in spaced-apart within the cavity, wherein a plurality of openings are provided between each pair of longitudinal nonmagnetic bars, To provide a flat surface for receiving and supporting the processing chamber, the upper surface is provided with a non-magnetic sheet that is in contact with the plurality of longitudinal non-magnetic bars, The system according to claim 12.

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