Method and device for producing car t cells

Abstract

The present invention relates to methods and devices for producing CAR T cells. A method of producing a population of T cells expressing a chimeric antigen receptor (CAR) comprises the steps of: providing a fluid sample comprising the population of T cells; marking the T cell population with magnetic beads; extracting a population of T cells from the fluid sample by flowing the fluid sample through a first conduit through a first magnetic separation device; transforming the T cell population to express a chimeric antigen receptor (CAR) by contacting the T cell population with a lentiviral population contained in a solution in the culture vessel; the population of T cells is collected by flowing the solution through a second conduit through a second magnetic separation device, wherein the first conduit, the culture vessel and the second conduit are fluidly connected by a fluid line network.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to provisional application No. 63 / 575,518, filed April 5, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the production of CAR T cells, and more particularly to methods and devices for producing CAR T cells. Background Art

[0004] Immunotherapy involving T cells engineered to express chimeric antigen receptors (CARs) has become an important treatment option for patients with B-cell lymphoma, acute lymphoblastic leukemia, or multiple myeloma. However, current methods for producing CAR T cells are complex and time-consuming, particularly for autologous treatment, where cell samples need to be transported back and forth between the treatment site and the cell production facility. There is a need to reduce the cost and time of CAR T cell production, particularly for autologous treatment. Summary of the Invention

[0005] The present invention relates to a method for meeting this need. The method for producing a T cell group expressing a chimeric antigen receptor (CAR) having the characteristics of the present invention comprises the following steps: providing a fluid sample comprising a T cell group; labeling the T cell group with magnetic beads; extracting the T cell group from the fluid sample by passing the fluid sample through a magnetic separation device; converting the T cell group to express a chimeric antigen receptor (CAR) by contacting the T cell group with a carrier carrying a CAR construct; and collecting the T cell group by passing the solution through a magnetic separation device or another magnetic separation device. The method may further include a step of activating the T cell group before converting the T cell group.

[0006] According to another aspect of the present invention, a method for producing a T cell population expressing a chimeric antigen receptor (CAR) comprises the following steps: providing a fluid sample comprising a T cell population; labeling the T cell population with magnetic beads; extracting the T cell population from the fluid sample by passing the fluid sample through a first conduit passing through a first magnetic separation device; contacting the T cell population with a lentivirus population contained in a culture container in a solution, transforming the T cell population to express a chimeric antigen receptor (CAR); collecting the T cell population by passing the solution through a second conduit passing through a second magnetic separation device, wherein the first conduit, the culture container, and the second conduit are fluidically connected by a fluid circuit network. The first conduit, the culture container, the second conduit, and the fluid circuit network are components of a closed fluid system. The method may further include the step of activating the T cell population before transforming the T cell population.

[0007] According to another aspect of the present invention, a method for producing a T cell population expressing a chimeric antigen receptor (CAR) comprises the following steps: providing a fluid sample comprising a T cell population; labeling the T cell population with magnetic beads; extracting the T cell population from the fluid sample by passing the fluid sample through a conduit passing through a magnetic separation device; contacting the T cell population with a lentivirus population contained in a culture container in a solution, transforming the T cell population to express a chimeric antigen receptor (CAR); collecting the T cell population by passing the solution through an acoustic separation device, wherein the conduit, culture container, and acoustic separation device are fluidically connected by a fluid circuit network. The conduit, culture container, acoustic separation device, and fluid circuit network are components of a closed fluid system. The method may further include the step of activating the T cell population before transforming the T cell population.

[0008] According to another aspect of the present invention, a method for producing a T cell population expressing a chimeric antigen receptor (CAR) comprises the following steps: providing a fluid sample comprising a T cell population and other cells; labeling the other cells with magnetic beads; removing the other cells from the fluid sample by passing the fluid sample through a conduit passing through a magnetic separation device; contacting the T cell population with a lentiviral population contained in a culture container in a solution, transforming the T cell population to express a chimeric antigen receptor (CAR); collecting the T cell population by passing the solution through an acoustic separation device, wherein the conduit, culture container, and acoustic separation device are fluidically connected by a fluid circuit network. The conduit, culture container, acoustic separation device, and fluid circuit network are components of a closed fluid system. The method may further include a step of activating the T cell population before transforming the T cell population.

[0009] According to another aspect of the present invention, a method for producing a T cell population expressing a chimeric antigen receptor (CAR) comprises the following steps: providing a fluid sample comprising a T cell population; labeling the T cell population with magnetic beads; extracting the T cell population from the fluid sample by passing the fluid sample through a first conduit passing through a first magnetic separation device; contacting the T cell population with a lentiviral population contained in a culture container and removing the magnetic beads from the surface of the T cells, transforming the T cell population to express a chimeric antigen receptor (CAR); removing the magnetic beads in the solution by passing the solution through a second conduit passing through a second magnetic separation device; collecting the T cell population by passing the solution through an acoustic separation device, wherein the first conduit, the culture container, the second conduit, and the acoustic separation device are fluidically connected by a fluid circuit network. The first conduit, the culture container, the second conduit, the acoustic separation device, and the fluid circuit network are components of a closed fluid system. The method may further include the step of activating the T cell population before transforming the T cell population. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings, in which:

[0011] Figure 1 is a block diagram illustrating an integrated system and its components that can be used to produce CAR T cells according to a first embodiment of the present invention;

[0012] Figure 2 is a front view of an integrated system for producing CAR T cells from whole blood or leukapheresis products according to a first embodiment of the present invention;

[0013] Figure 3 is a cross-sectional view corresponding to a column-less magnetic separation device for sorting magnetically labeled cells flowing through a catheter when the stent and catheter are detached from the magnetic assembly;

[0014] Figure 4 yes Figure 3 A cross-sectional view of a magnetic separation device when a catheter is squeezed against the tip of a magnetic assembly by a stent during magnetic separation;

[0015] Figure 5 The invention relates to a method for manufacturing CAR T cells using a closed fluid assembly (in the form of an integrated tubing set) according to a first embodiment of the present invention. Figure 2 perspective of integrated systems;

[0016] Figure 6 corresponds to Figure 5 Fluid circuit diagram of the integrated system;

[0017] Figure 7 is a block diagram illustrating an integrated system and its components that can be used to produce CAR T cells according to a second embodiment of the present invention;

[0018] Figures 8A-8E are various views of acoustic separation devices that can be used to sort cells based on their physical properties;

[0019] Figure 9 is a front view of an integrated system for producing CAR T cells from whole blood or leukapheresis products according to the first embodiment, the second embodiment, and the third embodiment of the present invention;

[0020] Figure 10 The invention relates to a method for manufacturing CAR T cells using a closed fluid assembly (in the form of an integrated tubing set) according to a second embodiment of the present invention. Figure 9 perspective of integrated systems;

[0021] Figure 11 corresponds to Figure 10 Fluid circuit diagram of the integrated system;

[0022] Figure 12 is a block diagram illustrating an integrated system and its components that can be used to produce CAR T cells according to a third embodiment of the present invention;

[0023] Figure 13 The invention relates to a method for manufacturing CAR T cells using a closed fluid assembly (in the form of an integrated tubing set) according to a third embodiment of the present invention. Figure 9 perspective of integrated systems;

[0024] Figure 14 corresponds to Figure 13 Fluid circuit diagram of the integrated system;

[0025] Figure 15 is a block diagram illustrating an integrated system and its components that can be used to produce CAR T cells according to a fourth embodiment of the present invention;

[0026] Figure 16 A method for manufacturing CAR T cells using a closed fluid assembly (in the form of an integrated tubing set) according to a fourth embodiment of the present invention Figure 9 perspective of integrated systems;

[0027] Figure 17 corresponds to Figure 16 Fluid circuit diagram of the integrated system;

[0028] Figure 18A and Figure 18B is a dot plot showing T cell purity from a leukapheresis product sample before and after the magnetic separation process;

[0029] Figure 19 is a dot plot showing T cell activation after 24 hours of incubation with CD3 / CD28 magnetic beads;

[0030] Figure 20A and Figure 20B is a dot plot of transduced T cells after magnetic collection.

[0031] For clarity and simplicity, like elements and components will be used with like reference numerals and numbers throughout the drawings, which are not necessarily drawn to scale. DETAILED DESCRIPTION

[0032] In the summary of the invention above and in the detailed description, claims, and drawings below, reference is made to specific features (including method steps) of the present invention. It should be understood that the disclosure of the invention in this specification includes all possible combinations of specific features. For example, when a specific feature is disclosed in the context of a particular aspect or embodiment of the invention, or in a particular claim, that feature may also be used in conjunction with other aspects and embodiments of the invention, as well as with the invention as a whole.

[0033] When reference is made herein to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously, unless the context excludes such possibility, and the method may include one or more additional steps before a defined step, between two defined steps, or after all defined steps, unless the context excludes such possibility.

[0034] As used herein, the term "biological object" includes cells, bacteria, viruses, molecules, particles (including RNA and DNA), cell clusters, bacterial clusters, molecular clusters, and particle clusters.

[0035] The term "biological sample" as used herein includes blood, body fluids, liquids and solids from tissues, bone marrow, hair, nails, bones, teeth, body secretions from any part of the body, or surface swabs from any part of the body. "Fluid sample" or "sample fluid" or "liquid sample" or "sample solution" can include biological samples in their original liquid form, samples in which biological objects are dissolved or dispersed in a buffer, or biological samples that have been dissociated from their original non-liquid form and dispersed in a buffer. A buffer is a liquid in which biological objects can be dissolved or dispersed without introducing contaminants or unwanted biological objects. Biological objects and biological samples can be derived from humans or animals. Biological objects can also be obtained from plants and the environment (including air, water and soil). Fluid samples can contain various types of magnetic or optical labels, or one or more chemical reagents added in various process steps.

[0036] As used herein, the term "sample flow rate" or "flow rate" is used to describe the volume of fluid flowing through a channel, conduit, fluid component, fluid path, or fluid circuit cross section per unit time.

[0037] In cell sorting and enrichment techniques, the population of target biological objects is referred to as "specific" objects of interest, while those separated but undesirable biological objects are referred to as "nonspecific." The term "purity" describes the frequency of the target or specific biological object of interest and is expressed as a percentage by dividing the number of target biological objects by the total number of all biological objects. The term "recovery rate" or "recovery rate" describes the efficiency of biological object sorting and is expressed as a percentage by dividing the number of target biological objects recovered after sorting by the number of target biological objects present in the initial sample.

[0038] As used herein, "at least" followed by a number refers to a range starting with that number, which may have an upper limit or no upper limit, depending on the defined variables. For example, "at least 1" means 1 or greater than 1. The term "at most" followed by a number refers to a range ending with that number, which may have a lower limit of 1 or 0, or no lower limit, depending on the defined variables. For example, "at most 4" means 4 or less than 4, and "at most 40%" means 40% or less than 40%. When a range is given in this specification as "first number to second number" or "first number - second number", this means that the lower limit of the range is the first number and the upper limit is the second number. For example, "25 to 100 nanometers" means that the lower limit of the range is 25 nanometers and the upper limit is 100 nanometers.

[0039] Directional terms, such as "front," "back," "upper," and "lower," may be used with reference to the orientation of the figures shown. Spatially relative terms, such as "below," "beneath," "under," "lower," "upper," and "above," may be used herein to describe the relationship of one element to another element (or elements) shown in the figures. Because articles and elements can be positioned in a variety of different orientations, these terms are intended for descriptive purposes and are in no way limiting of the invention unless the context precludes such a possibility.

[0040] When referring herein to a material AB consisting of an element A and an element B, the material AB may be an alloy, a compound, or a combination thereof, unless the context excludes such a possibility.

[0041] Provided herein are production devices and methods for manufacturing CAR T cells.

[0042] Embodiment 1 (First Embodiment): Positive Magnetic Separation and Positive Magnetic Purification

[0043] In this embodiment, a first magnetic separation device is used to separate or extract magnetically labeled target cells from an initial sample for genetic modification. This process may be performed in a cell culture incubator, and then a second magnetic separation device is used to purify the genetically modified target cells.

[0044] Figure 11 is a block diagram illustrating an integrated system 100 and its components that can be used to produce CAR T cells. The integrated system 100 has control electronics, a user interface, hardware, software, and firmware (not shown), and includes a first container or bag 102 for holding a sample containing target cells to be processed, a first magnetic separation device 104, a cell culture chamber 106, a second magnetic separation device 108, a second container or bag 112 containing a buffer or solution for eluting target cells from the first magnetic separation device 104, a culture component reservoir 114, a third container or bag 116 containing a buffer or solution for eluting target cells from the second magnetic separation device 108, a fourth container or bag 110 for receiving the final cell product, and fluid lines 118-130 providing fluid interconnection between the various components of the integrated system 100.

[0045] A first container or bag 102 contains a fluid sample containing magnetically labeled target cells 132 (e.g., T cells). The fluid sample can include whole blood, a leukapheresis product, PBMCs, and / or other leukapheresis products containing magnetically labeled target cells. The magnetic marker can attach to the target cells during a culture process within the same first container or bag 102.

[0046] The first magnetic separation device 104 is connected to the first container or bag 102 via a fluid line 118 and is used to extract magnetically labeled target cells from a fluid sample.

[0047] The second container or bag 112 is connected to the inlet of the first magnetic separation device 104 via a fluid line 120 and contains a buffer or solution for eluting the magnetically labeled target cells remaining in the first magnetic separation device 104 after the fluid sample passes through. The buffer or solution in the second container or bag 112 can include a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0048] The cell culture chamber 106 can be used for cell genetic engineering, cell modification, cell transduction or cell transfection. The cell culture chamber 106 includes a cell container 134 therein, which is connected to the outlet of the first magnetic separation device 104 by a fluid line 122. The cell container 134 contains target cells with magnetic labels, a buffer or solution for eluting cells from the first magnetic separation device 104, and optionally one or more buffers or solutions from a culture component library 114 connected to the cell container 134 by a fluid line 124. The cell culture chamber 106 can have one or more gas lines (e.g., CO2, not shown) connected thereto to provide an environment with a desired gas composition (e.g., 5.0% ± 0.1% CO2). The cell culture chamber 106 can also have a heating and cooling mechanism (not shown) that can maintain a constant temperature (e.g., 37.0 ± 0.1°C) indoors. In addition, the cell culture chamber 106 can further include an air circulation mechanism, such as a fan, to circulate air or other gas mixtures therein to make the temperature of the entire culture chamber 106 more uniform. The cell container 134 and its contents can be heated or cooled by convection from the surrounding environment in the cell culture chamber 106. The cell container 134 can also have porous or permeable portions, such as membranes and vents, that allow gas molecules from the surrounding environment to diffuse or flow, thereby exposing the contents of the cell container 134 to the air or other gas mixture within the cell culture chamber 106.

[0049] The culture component library 114 may include one or more of the following: culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection.

[0050] The cell culture chamber 106 may further include devices capable of inducing opening of the target cell membrane, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, thereby allowing genetic material to enter the target cell from the surrounding culture medium in the cell container 134. Genetic material can also be introduced into the target cell by droplet-based genetic material injection or by permeation of the target cell membrane.

[0051] The cell culture chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genetic material into target T cells to induce the growth of chimeric antigen receptors (CARs) on the cell surface.

[0052] The second magnetic separation device 108 is connected to the cell container 134 in the cell culture chamber 106 via the fluid line 126 and is used to extract target cells that still retain surface magnetic labels from the solution and / or culture medium mixture used in the cell container 134, and these cells have been genetically modified after transduction or transfection.

[0053] The third container or bag 116 is connected to the inlet of the second magnetic separation device 108 via a fluid line 128 and contains a buffer or solution for eluting the magnetically labeled target cells remaining in the second magnetic separation device 108 after the fluid sample passes through. The buffer or solution in the third container or bag 116 can include a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0054] A fourth container or bag 110 is connected to the outlet of the second magnetic separation device 108 via a fluid line 130 and contains genetically modified target cells (e.g., CAR T cells) in a solution that can be administered intravenously to a patient.

[0055] The network of fluid lines 118 - 130 , containers or bags 102 , 112 , 116 , 110 , cell container 134 , and conduits through the first and second magnetic separation devices 104 , 108 may be constructed, interconnected, and provided as a disposable integrated kit that may be sterile and sealed from the surrounding environment.

[0056] Continue to refer Figure 1 The process begins by providing a first container or bag 102 containing a first fluid sample having magnetically labeled target cells 132 (e.g., T cells). For a previously frozen sample, the first fluid sample can be prepared by first thawing the frozen sample and then extracting the target cells and other biological objects (if any) from the thawed sample fluid by centrifugation. The resulting target cells and other biological objects (if any) are resuspended in a buffer and filtered using a mesh size of 15 to 100 microns. A reagent containing a magnetic label is added to the filtered buffer containing the target cells to form the first fluid sample. Alternatively, the target cells can be magnetically labeled by an indirect process by first adding a reagent containing an intermediate link that attaches to the target cells and then adding a reagent containing a magnetic label that attaches to the intermediate link.

[0057] The first fluid sample flows through fluid line 118 into the inlet of the first magnetic separation device 104 for a first magnetic separation process. As the first fluid sample flows through the first magnetic separation device 104, the magnetically labeled target cells 132 are retained in the first magnetic separation device 104 by the magnetic field, while the depleted first fluid sample exits the first magnetic separation device 104 and enters a waste container or bag (not shown). After the first fluid sample has completely passed through the first magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is weakened or removed, and a first eluent, such as a buffer, in the second container or bag 112 flows through fluid line 120 into the first magnetic separation device 104, eluting the magnetically labeled target cells 132 and entering the cell container 134 through fluid line 122. In addition, one or more solutions from the culture component library 114, such as, but not limited to, culture medium, vectors for cell transduction or transfection, lentivirus for transduction, or buffers or solutions for non-viral transfection, are injected into the cell container 134 through fluid line 124 to modify the genetic structure of the magnetically labeled target cells 132. Target cells 132 (e.g., T cells) with magnetic labels can be transduced to express CAR by contacting with a vector carrying a CAR construct, including viral vectors such as lentiviruses and retroviruses, and non-viral vectors such as plasmids, lipid nanoparticles, and mRNA.

[0058] The transduction or transfection process is performed in the cell culture chamber 106, where the magnetically labeled target cells 132 are immersed in a transduction or transfection medium comprising a first eluent and one or more solutions from the culture component reservoir 114. During the transduction or transfection process, the temperature and gas composition within the cell culture chamber 106 can be controlled. For example, while modifying the genetic structure of the magnetically labeled target cells 132 in the cell container 134, the temperature and gas composition within the cell culture chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a duration ranging from 10 minutes to 72 hours.

[0059] After the transduction or transfection process is completed, a buffer for facilitating the subsequent magnetic separation process can be added from the culture component library 114 to the transduction or transfection medium in the cell container 134. The mixture of the transduction or transfection medium and the buffer for facilitating the magnetic separation process (if any) forms a second fluid sample containing magnetically labeled target cells carrying the modified gene structure.

[0060] The second fluid sample then flows through fluid line 126 into the inlet of the second magnetic separation device 108, where the magnetically labeled target cells carrying the modified gene construct are extracted through a second magnetic separation process. As the second fluid sample flows through the second magnetic separation device 108, the magnetically labeled target cells are retained in the second magnetic separation device 108 by the magnetic field, while the depleted second fluid sample exits the second magnetic separation device 108 and enters a waste container or bag (not shown). After the second fluid sample has completely passed through the second magnetic separation device 108, the magnetic field acting on the magnetically labeled target cells is weakened or removed, and a second eluent, such as a buffer, in the third container or bag 116 flows through fluid line 128 into the second magnetic separation device 108, eluting the magnetically labeled target cells and entering the fourth container or bag 110 through fluid line 130. After the second magnetic separation process is complete, the fourth container or bag 110 containing the magnetically labeled target cells carrying the modified gene construct can be separated from the rest of the integrated system 100 without exposing its contents to the atmosphere. The contents of the fourth container or bag 110 can be administered intravenously to the patient.

[0061] Figure 2 1 is a front view of an integrated system 100 that can be used to produce CAR T cells from whole blood or T cell-containing samples (e.g., leukapheresis products) according to the above process. The system 100 can be a closed processing system using closed fluidic components and can be deployed in facilities that lack a strict sterile or clean environment, such as treatment facilities, thereby alleviating many common logistical issues encountered in autologous treatment.

[0062] Figure 2 Automated production system 100 is shown, including a housing or enclosure structure 140, a first magnetic separation module 142A, a second magnetic separation module 142B, and a third magnetic separation module 142C residing in the enclosure structure 140, an incubation module 144, a sample rack 146 mounted on top of the enclosure structure 140, and a first sample bag rack 164 and a second sample bag rack 166. Fluid lines and sample bags connected thereto, which may be provided as integrated disposable tubing sets, and a computer for controlling system 100 are omitted from the figure to present an unobstructed view of magnetic separation modules 142A-142C.

[0063] The culture module 144 includes a culture chamber 106 located on top of a rocking base 147. Each magnetic separation module 142A-142C includes a first pinch valve 148A, 148B, 148C, a second pinch valve 150A, 150B, 150C, a third pinch valve 152A, 152B, 152C, a fourth pinch valve 154A, 154B, 154C, a first air detector 156A, 156B, 156C, a second air detector 158A, 158B, 158C, a peristaltic pump 160A, 160B, 160C, an occlusion sensor 162A, 162B, 162C, and a magnetic separation device 104, 105, 108.

[0064] The system 100 may further include additional pinch valves 168, 170 located on one side of the housing structure 140, and additional pinch valves 172, 174 located on the other side of the housing structure 140. The electromechanical assemblies 104-108, 147, 148A / B / C-162A / B / C, 168-174 may be controlled or automated by a computer or microprocessor (not shown).

[0065] Each magnetic separation device 104, 105, 108 may have the same or substantially similar structure as the magnetic separation device 188, and its cross-sectional view is as shown in FIG. Figure 3 . The magnetic separation device 188 includes a magnetic assembly 190 for generating a magnetic field, a conduit 192 made of a flexible and / or elastic material for flowing and sorting a fluid sample, and a bracket 194 for supporting the conduit 192. This figure shows that the conduit 192 and the bracket 194 are separated from the magnetic assembly 190. However, during the magnetic separation process, the conduit 192 will be placed close to the magnetic assembly 190, thereby exposing the conduit 192 to the magnetic field generated by the magnetic assembly 190.

[0066] The magnetic assembly 190 for generating a magnetic field to attract a biological object with a magnetic marker in a catheter 192 includes a magnetic flux source, which includes a first permanent magnet 193 and a second permanent magnet 195, a central magnetic flux guide 196 for conducting the magnetic flux of the magnetic flux source and forming a magnetic field, and a first side magnetic flux guide 198 and a second side magnetic flux guide 200 located on opposite sides of the central magnetic flux guide 196, which are used to conduct the magnetic flux of the magnetic flux source and form a magnetic field at the gap between the guides 196-200.

[0067] The center flux guide 196 has a tapered center tip 201 and a center base 203 that is physically and / or magnetically coupled to the first permanent magnet 193 and the second permanent magnet 195 at a first pole (e.g., north pole). The cross-section of the center tip 201 (defined herein as the cross-sectional area perpendicular to the flow of magnetic flux) can be smaller than the center base 203, thereby concentrating the magnetic flux from the center base 203 to the center tip 201. The first side flux guide 198 has a first side tip 202 and a first side base 204 that is physically and / or magnetically coupled to the first permanent magnet 193 at a second pole (e.g., south pole). The cross-section of the first side tip 202 can be smaller than the first side base 204, thereby concentrating the magnetic flux from the first side base 204 to the first side tip 202. The second side flux guide 200 has a second side tip 206 and a second side base 208 that is physically and / or magnetically coupled to the second permanent magnet 195 at the second pole (e.g., south pole). The cross-section of the second side tip 206 can be smaller than the second side base 208, thereby concentrating the magnetic flux from the second side base 208 to the second side tip 206. As a result, the magnetic flux density of each tip 201, 202, and 206 can be higher than the corresponding base 203, 204, or 208. The first side flux guide 198 and the second side flux guide 200 can be parallel at their bases 204 and 208 and curved or angled toward the center tip 201 at the tips 202 and 206, which can be pointed toward each other. The distal ends of the first side tip 202 and the second side tip 206 can each have a chiseled edge profile with a bevel facing upward or outward away from the center flux guide 196. The center tip 201 can be positioned below the first side tip 202 and the second side tip 206. During the magnetic sorting process, the catheter 192 can be operatively nested in a gap or concave space defined by the tip end of the center tip 201 and the bevels of the first side tip 202 and the second side tip 206, thereby exposing the catheter 192 to the magnetic field generated by the magnetic assembly 190.

[0068] The first permanent magnet 193 can be disposed between the center base 203 and the first side base 204, and the second permanent magnet 195 can be disposed between the center base 203 and the second side base 208. The first permanent magnet 193 and the second permanent magnet 195 have opposite magnetization directions and can be oriented substantially perpendicular to the center flux guide 196.

[0069] The center base 203 is magnetically coupled with the first and second permanent magnets 193 and 195 at their first poles (e.g., north poles), while the first and second side bases 204 and 208 are magnetically coupled with the first and second permanent magnets 193 and 195 at their second poles (e.g., south poles), respectively, such that the first and second side tips 202 and 206 (second polarity) and the center tip 201 (first polarity) have opposite magnetic polarities and strong magnetic fields are formed at or near the gaps between the tips 201, 202, and 206 to deposit magnetically labeled biological objects on the conduit wall.

[0070] With continued reference to Figure 3 The bracket 194 can have a first surface 210 facing the conduit 192 and a second surface 212 opposite the first surface 210. The first surface 210 can have a ridge structure 214 protruding from the first surface 210 that acts as a mechanical pressure device during the magnetic separation process to push the conduit 192 into the gap or concave space delineated by the tip end of the center tip 201 and the bevels of the first and second side tips 202 and 206. In addition, the ridge structure 214 of the bracket 194 can be made of a magnetically permeable material, such as a “floating” or top flux guide. In addition to acting as a mechanical pressure device to push the conduit 192 towards the tips 201, 202, and 206, the ridge structure 214 made of a magnetic material can magnetically interact with the tips 201, 202, and 206, further enhancing the magnetic field therebetween, thereby improving the magnetic separation efficiency.

[0071] Figure 4 is a cross-sectional view of the magnetic separation device 188 when the conduit 192 is squeezed by the ridge structure 214 of the bracket 194 and the tip ends 201, 202, and 206 of the three flux guides 196-200 during the magnetic separation process. The bracket 194 can further push the deformed or twisted conduit 192 into the gap between the center tip 201 and the first side tip 202 and the gap between the center tip 201 and the second side tip 206, where the magnetic field can be the strongest. Pushing the conduit 192 towards the tip end of the center tip 201 and the bevels of the first and second side tips 202 and 206 can expose more fluid samples flowing through the conduit 192 to a stronger magnetic field. During the magnetic separation process, magnetically labeled biological objects 216 can be deposited at the bottom of the conduit 192 near the center tip 201, where the magnetic field gradient can be the highest.

[0072] In embodiments where the ridge structure 214 is made of a soft magnetic material, it can act as a top magnetic flux guide when the ridge structure 214 is positioned close to the tips 201, 202, and 206 during magnetic sorting. The magnetic ridge structure 214 can conduct magnetic flux from the first side magnetic flux guide 198 and the second side magnetic flux guide 200, and thus can have the same magnetic polarity (second polarity) as the first side tip 202 and the second side tip 206, further enhancing the magnetic field between the ridge structure 214 and the center tip 201.

[0073] The magnetic flux guides 196-200 can each be made of a soft magnetic material or a material with a higher magnetic permeability, including iron (Fe), cobalt (Co), nickel (Ni), or any combination thereof. For example, but not by way of limitation, any of the magnetic flux guides 196-200 can be made of iron. The conduit 192 can be made of any suitable flexible and / or elastic material that can bend or deform, such as, but not limited to, rubber, plastic, or any suitable polymeric material. The holder 194 can be made of any suitable non-magnetic material, such as, but not limited to, aluminum, glass, a non-ferrous metal or alloy, plastic, or any suitable polymeric material. In some embodiments, the ridge structure 214 of the holder 194 that is in contact with the conduit 192 can be made of a soft magnetic material, such as, but not limited to, any of the soft magnetic materials described above for the magnetic flux guides 196-200.

[0074] Operation of the magnetic separation device 188, including recovery of magnetically labeled target cells, is described in greater detail in U.S. Patent Application Serial No. 18 / 795,047, which is incorporated by reference herein in its entirety. Other magnetic flux sources and column-free magnetic separation devices, such as those disclosed in U.S. Application Serial No. 18 / 072,362 (incorporated by reference herein), can also be used to isolate magnetically labeled target cells from a fluid sample.

[0075] Returning to Figure 2The incubation module 144 includes a cell culture chamber 106 on top of a rocking base 147. The culture chamber 106 has a heater that can uniformly maintain the temperature in the chamber between room temperature and 50°C. The C02concentration in the culture chamber 106 can be varied between 0% and 10% to provide the optimal environment for the cells. The rocking base 147 can move the culture chamber 106 at a speed of 2 to 4 revolutions per minute. The rocking motion of the culture chamber 106 caused by the rocking base 147 can promote uniform mixing of the fluid contents in the cell container 134. During cell activation, transduction, or expansion, the rocking base 147 can remain stationary. After the incubation process, the rocking base 147 can rock the cell container 134 within the culture chamber 106 to flush the cells in the cell container 134 off the surface of the container. The rocking base 147 can also be used to facilitate the drainage of the cell container 134 by tilting the culture chamber 106 to a fixed position with the end of the outlet tube reaching the bottom of the fluid in the cell container 134.

[0076] The system 100 has a modular design, including three magnetic separation modules 142A-142C and one incubation module 144. The modules 142A-142C, 144 can be fluidically connected in series, in parallel, or a combination thereof, by closed fluidic assemblies including sample bags, cell containers, and all fluidic lines, including conduits through the magnetic separation devices.

[0077] Figure 5 is a perspective view of the system 100 manufacturing CAR T cells using closed fluidic assemblies (in the form of integrated tube sets). For clarity, Figure 2 The electromechanical components labeled in Figure 2 Clamp valves 168, 170 on one side of the housing structure 140 in the front view. The lid and a portion of the side wall of the culture chamber 106 are removed to expose the cell container 134 in the form of a common cell culture flask within the culture chamber 106. The lid of the cell culture flask 134 contains an air filter that ventilates to the environment within the culture chamber 106 while maintaining the sterility of the flask 134. Figure 6 is a corresponding Figure 5 fluidic circuit diagram of the system 100 shown.

[0078] Reference is made to Figure 2 , Figure 5 and Figure 6The fluidic components of system 100 are arranged to use a first magnetic separation device 104 and a third magnetic separation device 105 fluidically coupled in parallel to extract magnetically labeled target cells (e.g., T cells) from a first fluid sample in a first magnetic separation process, and a second magnetic separation device 108 for a subsequent second magnetic separation process. Using two magnetic separation devices 104 and 105 for the first magnetic separation process enables system 100 to increase sorting throughput and accommodate larger volumes of first fluid samples, such as whole blood.

[0079] The first, second, third and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Sample bags 102 and 112 are both fluidically connected to the inlet of the first magnetic separation device 104 and the third magnetic separation device 105. In addition to sample bag 116, another sample bag 220 (which may also contain a cryopreservation solution, a storage medium, a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof) is also fluidically connected to the inlet of the second magnetic separation device 108. The culture component library 114 includes at least two sample bags 114A, 114B, which are fluidically connected to the cell container 134 through the magnetic separation devices 104, 105. Each sample bag 114A, 114B may include culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, or a cell activation reagent. Prior to the transduction or transfection process, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (eg, a solution containing CD3 / CD28 magnetic beads) from one of the sample bags 114A, 114B into the cell container 134 .

[0080] The fluidics assembly of system 100 further includes a sample bag 222 fluidically connected to the outlets of the first and third magnetic separation devices 104, 105 for collecting waste from the devices 104, 105, and another sample bag 224 fluidically connected to the outlet of the second magnetic separation device 108 for collecting waste from the device 108. The sample bags 222 for collecting waste from the first and third magnetic separation devices 104, 105 can be placed in a first bag rack 164. Sample bags 110, 224 for collecting the final cell product and waste from the second magnetic separation device 108, respectively, can be placed in a second bag rack 166. All other sample bags 102, 112, 114A, 114B, 116, 220, which provide materials for the manufacturing process, can be hung on a sample rack 146. The fluidics assembly of system 100 can include additional sample bags for various purposes. Each sample bag 102, 110, 112, 114A, 114B, 116, 220-224 can have an inlet port with a Luer connector connected thereto and an outlet port with a drip chamber. In addition, the flow of the inlet and outlet ports of each sample bag can be controlled or regulated by one or more manually tightened clamps attached thereto.

[0081] The fluidic components of the system 100 may further include syringes 226, 228, and 230. The syringes 226 and 228 may be fluidically connected to the inlet of the cell container 134 and may be used to extract a sample of the magnetically separated target cells after the first magnetic separation process, or to inject culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection, or cell activation reagents into the cell container 134. The syringe 230 may be fluidically connected to the outlet of the cell container 134 and may be used to extract a sample of the genetically modified target cells after the transduction or transfection process is completed.

[0082] Continue to refer Figure 6The fluidic circuit corresponding to system 100 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, and cell container 134, all interconnected by a network of fluidic lines that pass through electromechanical components such as pinch valves 148A-148C, 150A-150C, 152A-152C, 154A-154C, 168-172, air detectors 156A-156C, 158A-158C, occlusion sensors 162A-162C, peristaltic pumps 160A-160C, and magnetic separation devices 104, 105, 108. Sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, and the network of fluidic lines including conduits passing through magnetic separation devices 104, 105, 108 can be constructed, interconnected, sterilized, and provided as a disposable integrated kit. The electromechanical components of system 100 are located outside the tube set and thus do not come into contact with the fluids within the tube set.

[0083] First container or sample bag 102, which can contain target cells to be processed, is fluidically connected by one or more fluidic lines to the inlet of a conduit passing through first magnetic separation device 104, the fluidic lines passing through first pinch valve 148A, first air detector 156A, second air detector 158A, peristaltic pump 160A, and occlusion sensor 162A, and is also fluidically connected by one or more fluidic lines to the inlet of a conduit passing through third magnetic separation device 105, the fluidic lines passing through first pinch valve 148B, first air detector 156B, second air detector 158B, peristaltic pump 160B, and occlusion sensor 162B. Second container or sample bag 112, which can contain a buffer solution or media, is fluidically connected by one or more fluidic lines to the inlet of a conduit passing through first magnetic separation device 104, the fluidic lines passing through second pinch valve 150A, second air detector 158A, peristaltic pump 160A, and occlusion sensor 162A, and is also fluidically connected by one or more fluidic lines to the inlet of a conduit passing through third magnetic separation device 105, the fluidic lines passing through second pinch valve 150B, second air detector 158B, peristaltic pump 160B, and occlusion sensor 162B.

[0084] The inlet of the cell container 134 is fluidly connected by one or more fluid lines to the outlet of the conduit through the first magnetic separation device 104, the fluid lines passing through the third pinch valve 152A, and is also fluidly connected by one or more fluid lines to the outlet of the conduit through the third magnetic separation device 105, the fluid lines passing through the third pinch valve 152B. The syringes 226, 228 can be fluidly connected to the fluid lines between the inlet of the cell container 134 and the third pinch valves 152A, 152B. The sample bag 222, which can store waste fluid, e.g., sample fluid after target cells are extracted, is fluidly connected by one or more fluid lines to the outlet of the conduit through the first magnetic separation device 104, the fluid lines passing through the fourth pinch valve 154A, and is also fluidly connected by one or more fluid lines to the outlet of the conduit through the third magnetic separation device 105, the fluid lines passing through the fourth pinch valve 154B.

[0085] The outlet of the cell container 134 is fluidly connected by one or more fluid lines to the inlet of the conduit through the second magnetic separation device 108, the fluid lines passing through the first pinch valve 148C, the first air detector 156C, the second air detector 158C, the peristaltic pump 160C, and the occlusion sensor 162C. The syringe 230 can be fluidly connected to the fluid lines connected to the outlet of the cell container 134. The third container or sample bag 116 is fluidly connected by one or more fluid lines to the inlet of the conduit through the second magnetic separation device 108, the fluid lines passing through the pinch valve 172, the second pinch valve 150C, the second air detector 158C, the peristaltic pump 160C, and the occlusion sensor 162C. The sample bag 220 is fluidly connected by one or more fluid lines to the inlet of the conduit through the second magnetic separation device 108, the fluid lines passing through the pinch valve 174, the second pinch valve 150C, the second air detector 158C, the peristaltic pump 160C, and the occlusion sensor 162C.

[0086] The outlet of the conduit through the second magnetic separation device 108 is fluidly connected by one or more fluid lines to the fourth container or sample bag 110, the fluid lines passing through the third pinch valve 152C, and is also fluidly connected by one or more fluid lines to the sample bag 224, which can be used to receive waste fluid, the fluid lines passing through the fourth pinch valve 154C.

[0087] The sample bag 114A of the culture media bank 114 is fluidly connected to the inlet of the cell container 134 through one or more fluid lines through the pinch valve 168, the second pinch valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104, and the third pinch valve 152A, and through one or more fluid lines through the pinch valve 168, the second pinch valve 150B, the second air detector 158B, the peristaltic pump 160B, the occlusion sensor 162B, the third magnetic separation device 105, and the third pinch valve 152B. Similarly, the sample bag 114B of the culture media bank 114 is fluidly connected to the inlet of the cell container 134 through one or more fluid lines through the pinch valve 170, the second pinch valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104, and the third pinch valve 152A, and through one or more fluid lines through the pinch valve 170, the second pinch valve 150B, the second air detector 158B, the peristaltic pump 160B, the occlusion sensor 162B, the third magnetic separation device 105, and the third pinch valve 152B.

[0088] Embodiment 2 (Second Embodiment): Positive magnetic sorting and acoustic purification

[0089] This embodiment uses a magnetic separation device to isolate or extract target cells with magnetic labels for genetic modification, which can be performed in a cell incubator, and then uses an acoustic separation device to purify the genetically modified target cells.

[0090] Figure 7 is a block diagram showing an integrated system 300 that can be used to produce CAR T cells and components thereof. The integrated system 300 has control electronics, user interfaces, hardware, software, and firmware (not shown) including the first container or bag 102, the magnetic separation device 104, the cell culture chamber 106, the acoustic separation device 302, the second container or bag 112 containing a buffer or solution for eluting target cells from the magnetic separation device 104, the culture media bank 114, the third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, the fourth container or bag 110 for a final cell product, and a plurality of fluid lines providing fluid interconnections between the various components of the integrated system 300 through fluid lines 118-124 and 304-308. The integrated system 300 is similar to the integrated system 100 shown Figure 1 is a block diagram showing an integrated system 300 that can be used to produce CAR T cells and components thereof. The integrated system 300 has control electronics, user interfaces, hardware, software, and firmware (not shown) including the first container or bag 102, the magnetic separation device 104, the cell culture chamber 106, the acoustic separation device 302, the second container or bag 112 containing a buffer or solution for eluting target cells from the magnetic separation device 104, the culture media bank 114, the third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, the fourth container or bag 110 for a final cell product, and a plurality of fluid lines providing fluid interconnections between the various components of the integrated system 300 through fluid lines 118-124 and 304-308. The integrated system 300 is similar to the integrated system 100 shown

[0091] A first container or bag 102 contains a fluid sample containing magnetically labeled target cells 132 (e.g., T cells). The fluid sample can include whole blood, leukapheresis products, PBMCs, and / or other leukapheresis products containing magnetically labeled target cells 132. The magnetic markers can attach to the target cells 132 during a culture process within the same first container or bag 102.

[0092] The magnetic separation device 104 is connected to the first container or bag 102 via a fluid line 118 and is used to extract the target cells 132 with magnetic labels from the fluid sample. The magnetic separation device 104 can have the same or substantially similar structure as the magnetic separation device 188, and its cross-sectional view is shown in FIG. Figure 3 、 Figure 4 shown and described above.

[0093] The second container or bag 112 is connected to the inlet of the magnetic separation device 104 via a fluid line 120 and contains a buffer or solution for eluting the magnetically labeled target cells 132 remaining in the magnetic separation device 104 after the fluid sample passes through. The buffer or solution in the second container or bag 112 can include a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0094] The cell culture chamber 106 can be used for cell genetic engineering, cell modification, cell transduction or cell transfection. The cell culture chamber 106 includes a cell container 134 therein, which is connected to the outlet of the magnetic separation device 104 by a fluid line 122. The cell container 134 contains target cells 132 with magnetic labels, a buffer or solution for eluting cells from the magnetic separation device 104, and optionally one or more buffers or solutions from a culture component library 114 connected to the cell container 134 by a fluid line 124. The cell culture chamber 106 can have one or more gas lines (e.g., CO2, not shown) connected thereto to provide an environment with a desired gas composition (e.g., 5.0% ± 0.1% CO2). The cell culture chamber 106 can also have a heating and cooling mechanism (not shown) that can maintain a constant temperature (e.g., 37.0 ± 0.1°C) indoors. In addition, the cell culture chamber 106 can further include an air circulation mechanism, such as a fan, to circulate air or other gas mixtures therein to make the temperature of the entire culture chamber 106 more uniform. The cell container 134 and its contents can be heated or cooled by convection from the surrounding environment in the cell culture chamber 106. The cell container 134 can also have porous or permeable portions, such as membranes and vents, that allow gas molecules from the surrounding environment to diffuse or flow, thereby exposing the contents of the cell container 134 to the air or other gas mixture within the cell culture chamber 106.

[0095] The culture component library 114 may include one or more of the following: culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection.

[0096] The cell culture chamber 106 may further include devices capable of inducing opening of the target cell membrane, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, thereby allowing genetic material to enter the target cell from the surrounding culture medium in the cell container 134. Genetic material can also be introduced into the target cell by droplet-based genetic material injection or by permeation of the target cell membrane.

[0097] The cell culture chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genetic material into target cells to induce the growth of chimeric antigen receptors (CARs) on the cell surface.

[0098] The acoustic separation device 302 is connected to the cell container 134 in the cell culture chamber 106 via a fluid line 304 and is used to extract target cells from the solution and / or culture medium mixture used in the cell container 134, which cells have been genetically modified after transduction or transfection. Figures 8A-8E The same or substantially similar structure as the acoustic separation device 428 shown and described below.

[0099] Figure 8A and Figures 8B-8D 4 and 5. The figures are a top view and a corresponding cross-sectional view of an acoustic separation device 428, respectively, which is fabricated from a microfluidic chip 400. The microfluidic chip 400 includes a flat substrate 402 having a first flat surface 404 and a second flat surface 406 opposite the first flat surface 404, and a cover 408 attached to the first flat surface 404 of the flat substrate 402. The flat substrate 402 includes a channel network 411 recessed from the first flat surface 404 and substantially covered by the cover 408. The channel network 411 includes a separation channel 410 having an upstream end and a downstream end, a side inlet 412 for introducing a first fluid into the separation channel 410, two side inlet channels 414 connecting the side inlet 412 to a central inlet channel 418 at or near the upstream end of the separation channel 410, a central inlet 416 for introducing a second fluid into the separation channel 410, connecting the central inlet 416 to a central inlet channel 418 at or near the upstream end of the separation channel 410, a side outlet 420 for extracting a third fluid from the separation channel 410, a pair of side outlet channels 422 connecting the side outlet 420 to at or near the downstream end of the separation channel 410, a central outlet 424 for extracting a fourth fluid from the separation channel 410, and connecting the central outlet 424 to a central outlet channel 426 at or near the downstream end of the separation channel 410.

[0100] Figure 8B side and center inlets 412, 416 in the flat substrate 402 and Figure 8D the center outlet 424 in the flat substrate 402 opens to the outside of the microfluidic chip 400 on the second flat surface 406. However, any inlet or outlet 412, 416, 420, 424 can also open to the outside of the microfluidic chip 400 through the cover 408, which can include through-holes aligned with the respective port locations in the flat substrate 402.

[0101] Returning to Figure 8A , the separation channel 410 can have a linear shape with a nominal width W between its two side walls. W is in the range of about 100 micrometers to 1 millimeter. One or more of the side inlet channel 414, the center inlet channel 418, the side outlet channel 422, and the center outlet channel 426 can be narrower than the nominal width of the separation channel 410. Referring to Figure 8C , the separation channel 410 can have a nominal depth D measured from the first flat surface 404. D is in the range of about 50 micrometers to 500 micrometers. One or more of the side inlet channel 414, the center inlet channel 418, the side outlet channel 422, and the center outlet channel 426 can be shallower than the nominal depth of the separation channel 410.

[0102] A pair of side inlet channels 414 are connected to the separation channel 410 near or at its upstream end at its two side walls. Thus, a first fluid flowing through the pair of side inlet channels 414 is introduced into the separation channel 410 as two streams near the two side walls of the separation channel 410. The center inlet channel 418 is connected to the separation channel 410 at or near its center. A second fluid flowing through the center inlet channel 418 is introduced into the center of the separation channel 410 and pinched between the two streams of the first fluid at or near the upstream end of the separation channel 410.

[0103] A pair of side outlet channels 422 are connected to the separation channel 410 at or near its downstream end at its two side walls. Thus, the fluid flowing near the two side walls at or near the downstream end of the separation channel 410 is split by the pair of side outlet channels 422 into a third fluid and exits the microfluidic chip 400 through the side outlet 420. The remaining fluid not split by the pair of side outlet channels 422 at or near the center of the separation channel 410 becomes a fourth fluid and continues to flow through the center outlet channel 426 and exits the microfluidic chip 400 through the center outlet 424.

[0104] Figure 8EThe fluid paths through the microfluidic chip 400 according to the above-mentioned port positions are shown. The first and second fluids are introduced into the microfluidic chip 400 through the side inlet 412 and the center inlet 416, respectively. The third and fourth fluids are extracted from the microfluidic chip 400 through the side outlet 420 and the center outlet 424, respectively. The first fluid introduced through the side inlet 412 can be a fluid sample containing particles or biological objects of different sizes or acoustic contrasts, so as to be separated by acoustic radiation pressure, while the second fluid introduced through the center inlet 416 can be a buffer fluid without any particles or biological objects. After passing through the separation channel 410, the third fluid extracted from the side outlet 420 may include particles or biological objects of relatively small size or small acoustic contrast, while the fourth fluid extracted from the center outlet 424 may include particles or biological objects of relatively large size or high acoustic contrast.

[0105] The microfluidic chip 400 is part of an acoustic separation device 428, which further includes Figure 8E A piezoelectric transducer 430 is shown attached to the microfluidic chip 400. The piezoelectric transducer 430 can be attached to the second planar surface 406 of the planar substrate 402, opposite the separation channel 410. The piezoelectric transducer 430 can also be replaced by a cluster of two or more piezoelectric transducers distributed along the separation channel 410 and operating at the same frequency. The piezoelectric transducer 430 or the cluster of piezoelectric transducers can also be attached to the exterior of the lid 408 of the microfluidic chip 400. Alternatively, one or more piezoelectric transducers can be independently attached to the second planar surface 406 of the planar substrate 402 and the exterior of the lid 408.

[0106] The piezoelectric transducer 430 or a cluster of piezoelectric transducers can receive power in the form of an oscillating voltage with a frequency in the range of 100 kHz to 100 MHz to generate an acoustic pressure wave when liquid is present between the two sidewalls of the separation channel 410. When the channel width W is an integer multiple of half the wavelength of the acoustic pressure wave, an acoustic standing wave may form in the separation channel 410, depending on the excitation frequency applied to the piezoelectric transducer 430 and the compressibility and density of the liquid in the separation channel 410. When W is equal to half the wavelength of the acoustic wave generated by the piezoelectric transducer 430 or a cluster of piezoelectric transducers, a standing wave forms between the two sidewalls of the separation channel 410, with a single acoustic pressure node located at the center of the separation channel 410. Objects with relatively large size and / or high acoustic contrast may move toward the pressure node at a faster speed, thereby allowing objects to be separated by size or acoustic contrast.

[0107] Now refer to Figures 8A-8EThe operation of the acoustic separation device 428 under single pressure node conditions is described. After the transduction or transfection process is complete, a fluid sample from the cell container 134 (containing target cells 432 with modified genetic structures, small debris or unwanted biological objects 434 (i.e., non-target biological objects, if any), and transduction or transfection media) is introduced through the fluid line 304 into the side inlet 412, while a buffer fluid from the third container or bag 116 is introduced through the fluid line 306 into the center inlet 416. The target cells and unwanted biological objects 432 and 434 can have sufficiently different physical sizes and / or acoustic contrasts, such as mass density and compressibility, to allow separation by acoustic radiation pressure. For example, the target cells 432 can have a larger physical size and / or a higher acoustic contrast, such as a higher mass density and / or a lower compressibility, allowing acoustic radiation pressure to push the target cells 432 toward the pressure node located at the center of the separation channel 410 under single node conditions.

[0108] The fluid sample containing the target cells 432 and non-target biological objects 434 is introduced into the separation channel 410 through a pair of side inlet channels 414 at or near the upstream end of the separation channel 410 as two laminar flows 436 and 438 flowing close to the side walls. The two laminar flows 436 and 438 (possibly exhibiting laminar behavior) of the fluid sample in the separation channel 410 are separated by a central flow 440 of the buffer fluid from the center inlet 416. The central flow 440 of the buffer fluid can exhibit laminar behavior and acts as a sheath flow, slowing down or preventing the movement of the non-target biological objects 434 toward the pressure node located at the center of the separation channel 410. As the fluid sample progresses downstream along the separation channel 410, acoustic radiation pressure pushes the target cells 432 into the central flow 440 and toward the pressure node located at the center of the separation channel 410, while the non-target biological objects 434 mostly remain in the two laminar flows 436 and 438 close to the side walls. At the downstream end of the separation channel 410, the target cells 432 are carried by the buffer fluid out of the acoustic separation device 428 through the center outlet 424 into the fourth container or bag 110. The depleted fluid sample (possibly including the non-target biological objects 434) flows as laminar flows 436 and 438 close to the side walls and is diverted through a pair of side outlet channels 422 to the side outlet 420.

[0109] Referring back to Figure 7 , the third container or bag 116 is connected through the fluid line 306 to the inlet of the acoustic separation device 302 and contains a buffer or solution that can be used as a sheath flow in the acoustic separation device 302 when separating the genetically modified target cells. The buffer or solution can also be used as a storage solution for the genetically modified target cells in the fourth container or bag 110. The buffer or solution in the third container or bag 116 can include a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0110] A fourth container or bag 110 is connected to the outlet of the acoustic separation device 302 via a fluid line 308 and contains genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0111] The network of fluid lines 118-124 and 304-308, containers or bags 102, 110, 112, 116, cell container 134, and fluid lines through the magnetic separation device 104 and acoustic separation device 302 can be constructed, interconnected and provided as a disposable integrated tubing set that can be sterile and sealed from the surrounding environment.

[0112] Continue to refer Figure 7 The process begins by providing a first container or bag 102 containing a first fluid sample with magnetically labeled target cells 132. For a previously frozen sample, the first fluid sample can be prepared by first thawing the frozen sample and then extracting the target cells and other biological objects (if any) from the thawed sample fluid by centrifugation. The resulting target cells and other biological objects (if any) are resuspended in a buffer and filtered using a mesh size of 15 to 100 microns. A reagent containing a magnetic label is added to the filtered buffer containing the target cells to form the first fluid sample. Alternatively, the target cells can be magnetically labeled by an indirect process by first adding a reagent containing an intermediate link that attaches to the target cells and then adding a reagent containing a magnetic label attached to the intermediate link.

[0113] The first fluid sample flows through fluid line 118 into the inlet of the magnetic separation device 104 for magnetic separation (positive magnetic separation). As the first fluid sample flows through the magnetic separation device 104, the magnetically labeled target cells 132 are retained in the magnetic separation device 104 by the magnetic field, while the depleted first fluid sample exits the magnetic separation device 104 and enters a waste container or bag (not shown). After the first fluid sample has completely passed through the magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is weakened or removed, and the first eluent, such as a buffer, in the second container or bag 112 flows through fluid line 120 into the magnetic separation device 104, eluting the magnetically labeled target cells 132 into the cell container 134 through fluid line 122. In addition, one or more solutions from the culture component library 114, such as, but not limited to, culture medium, vectors for cell transduction or transfection, lentivirus for transduction, or buffers or solutions for non-viral transfection, are injected into the cell container 134 through fluid line 124 to modify the genetic structure of the magnetically labeled target cells 132. Target cells 132 (e.g., T cells) with magnetic labels can be transduced to express CAR by contacting with a vector carrying a CAR construct, including viral vectors such as lentiviruses and retroviruses, and non-viral vectors such as plasmids, lipid nanoparticles, and mRNA.

[0114] The transduction or transfection process is performed in the cell culture chamber 106, where the magnetically labeled target cells 132 are immersed in a transduction or transfection medium comprising a first eluent and one or more solutions from the culture component reservoir 114. During the transduction or transfection process, the temperature and gas composition within the cell culture chamber 106 can be controlled. For example, while modifying the genetic structure of the magnetically labeled target cells 132 in the cell container 134, the temperature and gas composition within the cell culture chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a duration ranging from 10 minutes to 72 hours.

[0115] After the transduction or transfection process is completed, a buffer for facilitating the subsequent acoustic sorting process can be added from the culture component library 114 to the transduction or transfection medium in the cell container 134. The mixture of the transduction or transfection medium and the buffer for facilitating the acoustic sorting process (if any) forms a second fluid sample containing magnetically labeled target cells carrying the modified gene structure.

[0116] Then, the second fluid sample flows into the inlet of the acoustic separation device 302 through the fluid line 304 to extract the target cells through the acoustic sorting process. Figures 8A-8ETaking the acoustic separation device 428 as an example, a second fluid sample containing genetically modified target cells 432 is introduced into the side inlet 412 through the fluid line 304, while a buffer is introduced into the central inlet 416 from the third container or bag 116 through the fluid line 306. The second fluid sample containing the genetically modified target cells 432 and non-target biological objects / debris 434 (if any) is introduced into the separation channel 410 near the upstream end of the separation channel 410 as two laminar flows 436 and 438 flowing close to the side walls through a pair of side inlet channels 414. The two laminar flows 436 and 438 of the second fluid sample in the separation channel 410 are separated by a central flow 440 of buffer from the central inlet 416. The central flow 440 of buffer can exhibit laminar behavior and act as a sheath flow, slowing or preventing the migration of non-target biological objects / debris 434 toward the pressure node located in the center of the separation channel 410. As the second fluid sample proceeds downstream along the separation channel 410, the acoustic radiation pressure pushes the genetically modified target cells 432 into the central flow 440 and toward the pressure node located in the center of the separation channel 410, while the non-target biological objects / debris 434 remain mostly within the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified target cells 432, carried by the buffer, exit the acoustic separation device 428 through the central outlet 424 and enter the fourth container or bag 110 through the fluid line 308. The further depleted second fluid sample, including the non-target biological objects / debris 434, flows near the side walls as laminar flows 436 and 438 and is diverted to the side outlet 420 through a pair of side outlet channels 422.

[0117] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified target cells can be separated from the rest of the integrated system 300 without exposing its contents to the atmosphere. The contents of the fourth container or bag 110 can be administered intravenously to a patient.

[0118] Figure 9 FIG3 is a front view of an integrated system 300 that can be used to produce CAR T cells from whole blood or T cell-containing samples according to the above process. System 300 can utilize closed fluidic components to form a closed processing system that can be deployed in facilities that lack a strict sterile or clean environment, such as treatment facilities, thereby alleviating many common logistical issues encountered in autologous treatments.

[0119] Figure 9Automated production system 300 is shown, including a housing or enclosure structure 140, first and second magnetic separation modules 142A, 142B residing in enclosure structure 140, and acoustic separation module 310, an incubation module 144, a sample rack 146 mounted on top of enclosure structure 140, and first and second sample bag racks 164, 166. Fluidic lines and sample bags connected thereto, which may be provided as integrated disposable tubing sets, and a computer for controlling system 300 are omitted from the figure to provide an unobstructed view of magnetic separation modules 142A, 142B and acoustic separation module 310. Production system 300 is similar to systems 100 and 500, except that the routing of the fluidic lines is different, as described below.

[0120] The culture module 144 includes a culture chamber 106 located on top of a rocking base 147. Each magnetic separation module 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and magnetic separation devices 104, 105. The acoustic separation module 310 includes a first peristaltic pump 312, a second peristaltic pump 314, a first occlusion sensor 316, a second occlusion sensor 318, a first air detector 320, a second air detector 322, an acoustic separation device 302, and first, second, and third pinch valves 324-328.

[0121] The system 300 may further include additional pinch valves 168, 170 located on one side of the housing structure 140, and additional pinch valves 172, 174 located on the other side of the housing structure 140. The electromechanical assemblies 104-106, 147, 148A / B-162A / B, 168-174, 312-328 may be controlled or automated by a computer or microprocessor (not shown).

[0122] Each magnetic separation device 104, 105 may have Figure 3 and 4 The same or substantially similar structure as the magnetic separation device 188 shown and described above. Other magnetic flux sources and pillarless magnetic separation devices, such as those disclosed in U.S. application Ser. No. 18 / 072,362 (incorporated herein by reference), can also be used to separate magnetically labeled target cells from a fluid sample.

[0123] The acoustic separation device 302 may have Figures 8A-8E The acoustic separation device 428 shown and described above may have the same or substantially similar structure.

[0124] The culture module 144 includes a cell culture chamber 106 atop a rocking base 147. The culture chamber 106 is equipped with a heater to maintain a uniform temperature between room temperature and 50°C. The CO2 concentration within the culture chamber 106 can be varied between 0% and 10% to provide an optimal environment for the cells. The rocking base 147 can move the culture chamber 106 at a speed of 2 to 4 revolutions per minute. The rocking motion of the culture chamber 106 caused by the rocking base 147 promotes uniform mixing of the fluid contents within the cell container 134. During cell activation, transduction, or expansion, the rocking base 147 can remain stationary. After the culture process, the rocking base 147 can rock the cell container 134 within the culture chamber 106, flushing the fluid within the cell container 134 from the surface of the container. The rocking base 147 can also be used to facilitate drainage of the cell container 134 by tilting the culture chamber 106 into a fixed position, allowing the end of the outlet tube to reach the bottom of the fluid within the cell container 134.

[0125] The system 300 has a modular design and includes two magnetic separation modules 142A, 142B, an acoustic separation module 310, and an incubation module 144. The modules 142A, 142B, 144, and 310 can be fluidically connected in series, in parallel, or a combination thereof via a closed fluidic assembly that includes sample bags, cell containers, an acoustic separation device, and all fluid lines (including a conduit passing through the magnetic separation device).

[0126] Figure 10 is a perspective view of a system 300 for manufacturing CAR T cells using a closed fluidic assembly in the form of an integrated tubing set. For clarity, Figure 9 The electromechanical components labeled in FIG are not relabeled in this figure to clearly illustrate the path of the fluid circuit. The perspective view also shows the pinch valves 152B, 154A, 154B previously obscured by the cell culture chamber 106, and Figure 9 Pinch valves 172, 174 are shown on one side of the housing structure 140 in a front view. The lid and a portion of the sidewall of the culture chamber 106 are removed to expose the cell container 134, which is in the form of a conventional cell culture flask, within the culture chamber 106. The lid of the cell culture flask 134 includes an air filter that vents air into the environment within the culture chamber 106 while maintaining the sterility of the flask 134. The second bag rack 166 is also omitted from the figure to illustrate the sample bags 110, 224 within the rack 166. Figure 11 corresponds to Figure 10 A fluid circuit diagram of system 300 is shown.

[0127] refer to Figure 9 、 Figure 10 、 Figure 11The fluidic components of system 300 are arranged to use a first magnetic separation device 104 and a second magnetic separation device 105 in parallel fluidic coupling to extract magnetically labeled target cells (e.g., T cells) from a first fluid sample during a magnetic sorting process (positive magnetic sorting), and an acoustic separation device 302 for a subsequent acoustic sorting process. Using two magnetic separation devices 104, 105 for the magnetic sorting process enables system 300 to increase sorting throughput and accommodate larger volumes of first fluid samples, such as whole blood.

[0128] The first, second, third and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Sample bags 102 and 112 are both fluidically connected to the inlet of the first magnetic separation device 104 and the second magnetic separation device 105. In addition to sample bag 116, another sample bag 220 (which may also contain a cryopreservation solution, a storage medium, a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof) is also fluidically connected to the inlet of the acoustic separation device 302. The culture component library 114 includes at least two sample bags 114A, 114B, which are fluidically connected to the cell container 134 through the magnetic separation devices 104, 105. Each sample bag 114A, 114B may include culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, or a cell activation reagent. Prior to the transduction or transfection process, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (eg, a solution containing CD3 / CD28 magnetic beads) from one of the sample bags 114A, 114B into the cell container 134 .

[0129] The fluidics assembly of system 300 further includes a sample bag 222 fluidically connected to the outlets of the first and second magnetic separation devices 104, 105 for collecting waste from the devices 104, 105, and another sample bag 224 fluidically connected to the outlet of the acoustic separation device 302 for collecting waste from the device 302. The sample bags 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in a first bag rack 164. Sample bags 110, 224 for collecting the final cell product and waste from the acoustic separation device 302, respectively, can be placed in a second bag rack 166. All other sample bags 102, 112, 114A, 114B, 116, 220, which provide materials for the manufacturing process, can be hung on the sample rack 146. The fluidics assembly of system 300 can include additional sample bags for various purposes. Each sample bag 102, 110, 112, 114A, 114B, 116, 220-224 can have an inlet port with a Luer connector connected thereto and an outlet port with a drip chamber. In addition, the flow of the inlet and outlet ports of each sample bag can be controlled or regulated by one or more manually tightened clamps attached thereto.

[0130] The fluidic components of the system 300 may further include syringes 226, 228, and 230. The syringes 226 and 228 may be fluidically connected to the inlet of the cell container 134 and may be used to extract a sample of the magnetically separated target cells after the magnetic separation process, or to inject culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, or a cell activation reagent into the cell container 134. The syringe 230 may be fluidically connected to the outlet of the cell container 134 and may be used to extract a sample of the genetically modified target cells after the transduction or transfection process is completed.

[0131] Continue to refer Figure 11, the fluid circuit corresponding to system 300 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134 and acoustic separation device 302, all of which are interconnected by a fluid line network that passes through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separation devices 104, 105. The sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, acoustic separation device 302, and a network of fluid lines including conduits passing through magnetic separation devices 104 and 105 can be constructed, interconnected, sterilized, and provided as a disposable integrated kit. The electromechanical components of system 300 are located outside the tubing set and therefore do not come into contact with the fluid within the tubing set.

[0132] The first container or sample bag 102, which may contain target cells to be processed, is fluidly connected to the inlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines, which pass through the first pinch valve 148A, the first air detector 156A, the second air detector 158A, the peristaltic pump 160A and the occlusion sensor 162A, and is also fluidly connected to the inlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines, which pass through the first pinch valve 148B, the first air detector 156B, the second air detector 158B, the peristaltic pump 160B and the occlusion sensor 162B. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines, which pass through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines, which pass through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B, and an occlusion sensor 162B.

[0133] The inlet of cell container 134 is fluidically connected to the outlet of the conduit passing through first magnetic separation device 104 via one or more fluid lines, which pass through third pinch valve 152A, and is also fluidically connected to the outlet of the conduit passing through second magnetic separation device 105 via one or more fluid lines, which pass through third pinch valve 152B. Syringes 226 and 228 can be fluidically connected to the fluid lines between the inlet of cell container 134 and third pinch valves 152A and 152B. Sample bag 222, which can store waste fluid, such as sample fluid after extraction of target cells, is fluidically connected to the outlet of the conduit passing through first magnetic separation device 104 via one or more fluid lines, which pass through fourth pinch valve 154A, and is also fluidically connected to the outlet of the conduit passing through second magnetic separation device 105 via one or more fluid lines, which pass through fourth pinch valve 154B.

[0134] The outlet of the cell container 134 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., Figure 8A The fluid lines pass through the first peristaltic pump 312, the first occlusion sensor 316, and the first air detector 320. The syringe 230 can be fluidically connected to the fluid line at the outlet of the cell container 134. The third container or sample bag 116 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., Figure 8A The sample bag 220 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., the central inlet 416 of the sample bag 220), which passes through the pinch valve 172, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324. Figure 8A ), these fluid lines pass through the pinch valve 174 , the second peristaltic pump 314 , the second occlusion sensor 318 and the first pinch valve 324 .

[0135] The fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic separation device 302 (eg, Figure 8A The sample bag 224, which can be used to receive waste fluid, is fluidically connected to the outlet of the acoustic separation device 302 (e.g., the central outlet 424 of the acoustic separation device 302) via one or more fluid lines. Figure 8A side outlet 420 ), these fluid lines pass through the second air detector 322 and the third pinch valve 328 .

[0136] The sample bag 114A of the culture component library 114 is fluidly connected to the inlet of the cell container 134 through one or more fluid lines, which pass through the clamping valve 168, the second clamping valve 150A, the second air detector 158A, the peristaltic pump 160A, the blockage sensor 162A, the first magnetic separation device 104 and the third clamping valve 152A, and through one or more fluid lines through the clamping valve 168, the second clamping valve 150B, the second air detector 158B, the peristaltic pump 160B, the blockage sensor 162B, the second magnetic separation device 105 and the third clamping valve 152B. Similarly, the sample bag 114B of the culture component library 114 is fluidly connected to the inlet of the cell container 134 through one or more fluid lines, which pass through the clamping valve 170, the second clamping valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104 and the third clamping valve 152A, and through one or more fluid lines through the clamping valve 170, the second clamping valve 150B, the second air detector 158B, the peristaltic pump 160B, the occlusion sensor 162B, the second magnetic separation device 105 and the third clamping valve 152B.

[0137] Embodiment 3 (Third embodiment): Negative magnetic separation and acoustic purification

[0138] This embodiment uses a magnetic separation device to remove magnetically labeled non-target cells, retaining the target T cells for genetic modification. This process may be performed in a cell culture incubator, and then an acoustic separation device is used to purify the genetically modified target cells.

[0139] Figure 12 1 is a block diagram illustrating an integrated system 500 and its components that can be used to produce CAR T cells. The integrated system 500 has control electronics, user interfaces, hardware, software, and firmware (not shown), and includes a first container or bag 102, a magnetic separation device 104, a cell culture chamber 106, an acoustic separation device 302, a second container or bag 112 containing a buffer or solution for eluting target cells from the magnetic separation device 104, a culture component reservoir 114, a third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, a fourth container or bag 110 for the final cell product, and a plurality of fluid lines 118-124, 306, 308 providing fluidic interconnections between the various components of the integrated system 500.

[0140] The first container or bag 102 contains a fluid sample containing a T cell population and other cells. The fluid sample can include whole blood, a leukapheresis product, PBMCs, and / or other leukapheresis products containing T cells. In this embodiment, non-target cells (i.e., cells other than T cells) are magnetically labeled during the culture process within the same first container or bag 102, rather than labeling T cells.

[0141] The magnetic separation device 104 is connected to the first container or bag 102 via a fluid line 118 and is used to remove non-target cells with magnetic labels from the fluid sample, thereby retaining unlabeled T cells. Figure 3 、 Figure 4 The magnetic separation device 188 shown and described above has the same or substantially similar structure.

[0142] The second container or bag 112 is connected to the inlet of the magnetic separation device 104 via a fluid line 120 and contains a buffer or solution for eluting unlabeled T cells remaining in the magnetic separation device 104 after the fluid sample passes through. The buffer or solution in the second container or bag 112 can include a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0143] The cell culture chamber 106 can be used for cell genetic engineering, cell modification, cell transduction or cell transfection. The cell culture chamber 106 includes a cell container 134 therein, which is connected to the outlet of the magnetic separation device 104 via a fluid line 122. The cell container 134 contains unlabeled T cells, a buffer or solution for eluting cells from the magnetic separation device 104, and optionally one or more buffers or solutions from a culture component library 114 connected to the cell container 134 via a fluid line 124. The cell culture chamber 106 can have one or more gas lines (e.g., CO2, not shown) connected thereto to provide an environment with a desired gas composition (e.g., 5.0% ± 0.1% CO2). The cell culture chamber 106 can also have a heating and cooling mechanism (not shown) that can maintain a constant temperature (e.g., 37.0 ± 0.1°C) indoors. In addition, the cell culture chamber 106 can further include an air circulation mechanism, such as a fan, to circulate air or other gas mixtures therein to make the temperature of the entire culture chamber 106 more uniform. The cell container 134 and its contents can be heated or cooled by convection from the surrounding environment in the cell culture chamber 106. The cell container 134 can also have porous or permeable portions, such as membranes and vents, that allow gas molecules from the surrounding environment to diffuse or flow, thereby exposing the contents of the cell container 134 to the air or other gas mixture within the cell culture chamber 106.

[0144] The culture component library 114 may include one or more of the following: culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection.

[0145] The cell culture chamber 106 may further include devices capable of inducing opening of the target cell membrane, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, thereby allowing genetic material to enter the target cell from the surrounding culture medium in the cell container 134. Genetic material can also be introduced into the target cell by droplet-based genetic material injection or by permeation of the target cell membrane.

[0146] The cell culture chamber 106 can provide an environment for the transduction process. For example, lentivirus or other viruses can be used to transfer genetic material into target cells to induce the growth of chimeric antigen receptors (CARs) on the cell surface. The acoustic separation device 302 is connected to the cell container 134 in the cell culture chamber 106 via a fluid line 304 to extract the genetically modified target cells from the solution and / or culture medium mixture used in the cell container 134.

[0147] The acoustic separation device 302 may have Figures 8A-8E The acoustic separation device 428 shown and described below has the same or substantially similar structure (not fully described here due to document truncation, and will be supplemented later).

[0148] The third container or bag 116 is connected to the inlet of the acoustic separation device 302 via a fluid line 306 and contains a buffer or solution that may be used as a sheath fluid when acoustically separating the genetically modified target cells in the acoustic separation device 302. The buffer or solution may also be used as a storage solution for preserving the genetically modified target cells in the fourth container or bag 110. The buffer or solution in the third container or bag 116 may include a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0149] A fourth container or bag 110 is connected to the outlet of the acoustic separation device 302 via a fluid line 308 and contains genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0150] The network of fluid lines 118-124, 306, 308, containers or bags 102, 110, 112, 116, cell container 134 and fluid lines through the magnetic separation device 104 and acoustic separation device 302 can be constructed, interconnected and provided as a disposable integrated tubing set that can be sterile and sealed from the surrounding environment.

[0151] Continue to refer Figure 12The process begins by providing a first container or bag 102 containing a first fluid sample of a T cell population and other cells. For a previously frozen sample, the first fluid sample can be prepared by first thawing the frozen sample and then extracting the cells from the thawed sample fluid by centrifugation. The resulting cells are resuspended in a buffer and filtered using a mesh size of 15 to 100 microns. A reagent containing a magnetic marker is added to the filter buffer containing the cells to mark non-target cells (i.e., cells other than T cells), thereby forming a first fluid sample.

[0152] The first fluid sample flows through fluid line 118 into the inlet of the magnetic separation device 104 for magnetic separation. When the first fluid sample flows through the magnetic separation device 104, the non-target cells with magnetic labels are retained in the magnetic separation device 104 by the magnetic field, while the depleted first fluid sample containing unlabeled T cells flows through fluid line 122 into the cell container 134. The buffer or solution in the second container or bag 112 flows into the magnetic separation device 104 through fluid line 120 to elute any remaining unlabeled T cells into the cell container 134. In addition, one or more solutions from the culture component library 114, such as but not limited to culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffer or solution for non-viral transfection, are injected into the cell container 134 through fluid line 124 to modify the genetic structure of unlabeled T cells. Unlabeled T cells can be transduced to express CAR by contacting with a vector carrying a CAR construct, including viral vectors such as lentivirus and retrovirus, and non-viral vectors such as plasmids, lipid nanoparticles and mRNA.

[0153] The transduction or transfection process is performed in the cell culture chamber 106, where unlabeled T cells are immersed in a transduction or transfection medium comprising a buffer and one or more solutions from the culture component reservoir 114. During the transduction or transfection process, the temperature and gas composition within the cell culture chamber 106 can be controlled. For example, when modifying the genetic structure of T cells in the cell container 134, the temperature and gas composition within the cell culture chamber 106 can be maintained at 37.0 ± 0.1°C and 5.0% ± 0.1% CO2, respectively, for a duration ranging from 10 minutes to 72 hours.

[0154] After the transduction or transfection process is completed, a buffer for facilitating the subsequent acoustic sorting process can be added from the culture component library 114 to the transduction or transfection medium in the cell container 134. The mixture of the transduction or transfection medium and the buffer for facilitating the acoustic sorting process (if any) forms a second fluid sample containing genetically modified T cells.

[0155] Then, the second fluid sample flows into the inlet of the acoustic separation device 302 through the fluid line 304 to extract the gene-modified T cells through the acoustic sorting process. Figures 8A-8E Taking the acoustic separation device 428 as an example, a second fluid sample containing genetically modified T cells is introduced into the side inlet 412 through the fluid line 304, while a buffer is introduced into the central inlet 416 from the third container or bag 116 through the fluid line 306. The second fluid sample containing the genetically modified T cells and non-target biological objects / debris (if any) is introduced into the separation channel 410 near the upstream end of the separation channel 410 through a pair of side inlet channels 414 as two laminar flows 436 and 438 flowing close to the side walls. The two laminar flows 436 and 438 of the second fluid sample in the separation channel 410 are separated by a central flow 440 of buffer from the central inlet 416. The central flow 440 of buffer can exhibit laminar behavior and act as a sheath flow, slowing or preventing the migration of non-target biological objects / debris toward the pressure node located in the center of the separation channel 410. As the second fluid sample progresses downstream along the separation channel 410, the acoustic radiation pressure pushes the genetically modified T cells into the central stream 440 and toward the pressure node located in the center of the separation channel 410, while the non-target biological objects / debris mostly remain in the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified T cells, carried by the buffer, exit the acoustic separation device 428 through the central outlet 424 and enter the fourth container or bag 110 through the fluid line 308. The further depleted second fluid sample, including the non-target biological objects / debris, flows as laminar flows 436 and 438 near the side walls and is diverted to the side outlet 420 through a pair of side outlet channels 422.

[0156] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified T cells can be separated from the rest of the integrated system 500 without exposing its contents to the atmosphere. The contents of the fourth container or bag 110 can be administered intravenously to a patient.

[0157] Figure 9 FIG5 is a front view of an integrated system 500 that can be used to produce CAR T cells from whole blood or T cell-containing samples according to the above process. System 500 can utilize closed fluidic components to form a closed processing system that can be deployed in facilities that lack a strict sterile or clean environment, such as treatment facilities, thereby alleviating many common logistical issues encountered in autologous treatments.

[0158] Figure 9An automated production system 500 is shown, comprising a housing or enclosure structure 140, first and second magnetic separation modules 142A, 142B, and acoustic separation module 310 residing within the enclosure structure 140, an incubation module 144, a sample rack 146 mounted atop the enclosure structure 140, and first and second sample bag racks 164, 166. Fluidic lines and sample bags connected thereto, which may be provided as integrated disposable tubing sets, and a computer for controlling the system 500 are omitted from the figure to provide an unobstructed view of the magnetic separation modules 142A, 142B and the acoustic separation module 310. Production system 500 is similar to system 300, except that the fluidic lines are routed differently to accommodate the negative magnetic separation process described below.

[0159] The culture module 144 includes a culture chamber 106 located on top of a rocking base 147. Each magnetic separation module 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and magnetic separation devices 104, 105. The acoustic separation module 310 includes a first peristaltic pump 312, a second peristaltic pump 314, a first occlusion sensor 316, a second occlusion sensor 318, a first air detector 320, a second air detector 322, an acoustic separation device 302, and first, second, and third pinch valves 324-328.

[0160] The system 500 may further include additional pinch valves 168, 170 located on one side of the housing structure 140, and additional pinch valves 172, 174 located on the other side of the housing structure 140. The electromechanical assemblies 104-106, 147, 148A / B-162A / B, 168-174, 312-328 may be controlled or automated by a computer or microprocessor (not shown).

[0161] Each magnetic separation device 104, 105 may have Figure 3 and 4 The same or substantially similar structure as the magnetic separation device 188 shown and described above. Other magnetic flux sources and pillarless magnetic separation devices, such as those disclosed in U.S. application Ser. No. 18 / 072,362 (incorporated herein by reference), can also be used to separate magnetically labeled target cells from a fluid sample.

[0162] The acoustic separation device 302 may have Figures 8A-8E The acoustic separation device 428 shown and described above may have the same or substantially similar structure.

[0163] The culture module 144 includes a cell culture chamber 106 atop a rocking base 147. The culture chamber 106 is equipped with a heater to maintain a uniform temperature between room temperature and 50°C. The CO2 concentration within the culture chamber 106 can be varied between 0% and 10% to provide an optimal environment for the cells. The rocking base 147 can move the culture chamber 106 at a speed of 2 to 4 revolutions per minute. The rocking motion of the culture chamber 106 caused by the rocking base 147 promotes uniform mixing of the fluid contents within the cell container 134. During cell activation, transduction, or expansion, the rocking base 147 can remain stationary. After the culture process, the rocking base 147 can rock the cell container 134 within the culture chamber 106, flushing the fluid within the cell container 134 from the surface of the container. The rocking base 147 can also be used to facilitate drainage of the cell container 134 by tilting the culture chamber 106 into a fixed position, allowing the end of the outlet tube to reach the bottom of the fluid within the cell container 134.

[0164] The system 500 has a modular design and includes two magnetic separation modules 142A, 142B, one acoustic separation module 310, and one incubation module 144. The modules 142A, 142B, 144, and 310 can be fluidically connected in series, in parallel, or a combination thereof through a closed fluidic assembly that includes sample bags, cell containers, an acoustic separation device, and all fluid lines (including a conduit passing through the magnetic separation device).

[0165] Figure 13 is a perspective view of a system 500 for manufacturing CAR T cells using a closed fluidic assembly in the form of an integrated tubing set. For clarity, Figure 9 The electromechanical components labeled in FIG are not relabeled in this figure to clearly illustrate the path of the fluid circuit. The perspective view also shows the pinch valves 152B, 154A, 154B, 328, which were previously obscured by the cell culture chamber 106, and Figure 9 Pinch valves 172, 174 are shown on one side of the housing structure 140 in a front view. The lid and a portion of the sidewall of the culture chamber 106 are removed to expose the cell container 134, which is in the form of a conventional cell culture flask, within the culture chamber 106. The lid of the cell culture flask 134 includes an air filter that vents air into the environment within the culture chamber 106 while maintaining the sterility of the flask 134. The second bag rack 166 is also omitted from the figure to illustrate the sample bags 110, 224 within the rack 166. Figure 14 corresponds to Figure 13 A fluid circuit diagram of system 500 is shown.

[0166] refer to Figure 9 、 1314. The fluidic components of system 500 are arranged to utilize a first magnetic separation device 104 and a second magnetic separation device 105 fluidically coupled in parallel to extract magnetically labeled non-target cells from a first fluid sample during a magnetic sorting process (negative magnetic sorting), and an acoustic separation device 302 for purifying non-magnetic target cells. Using two magnetic separation devices 104 and 105 for the magnetic sorting process enables system 500 to increase sorting throughput and accommodate larger volumes of first fluid samples.

[0167] The first, second, third and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Sample bags 102 and 112 are both fluidically connected to the inlet of the first magnetic separation device 104 and the second magnetic separation device 105. In addition to sample bag 116, another sample bag 220 (which may also contain a cryopreservation solution, a storage medium, a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof) is also fluidically connected to the inlet of the acoustic separation device 302. The culture component library 114 includes at least two sample bags 114A, 114B, which are fluidically connected to the cell container 134 through the magnetic separation devices 104, 105. Each sample bag 114A, 114B may include culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, or a cell activation reagent. Prior to the transduction or transfection process, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (eg, a solution containing CD3 / CD28 magnetic beads) from one of the sample bags 114A, 114B into the cell container 134 .

[0168] The fluidics assembly of system 500 further includes a sample bag 222 fluidically connected to the outlets of the first and second magnetic separation devices 104, 105 for collecting waste from the devices 104, 105, and another sample bag 224 fluidically connected to the outlet of the acoustic separation device 302 for collecting waste from the device 302. The sample bags 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in a first bag rack 164. Sample bags 110, 224 for collecting the final cell product and waste from the acoustic separation device 302, respectively, can be placed in a second bag rack 166. All other sample bags 102, 112, 114A, 114B, 116, 220, which provide materials for the manufacturing process, can be hung on the sample rack 146. The fluidics assembly of system 500 can include additional sample bags for various purposes. Each sample bag 102, 110, 112, 114A, 114B, 116, 220-224 can have an inlet port with a Luer connector connected thereto and an outlet port with a drip chamber. In addition, the flow of the inlet and outlet ports of each sample bag can be controlled or regulated by one or more manually tightened clamps attached thereto.

[0169] The fluidic components of the system 500 may further include syringes 226, 228, and 230. The syringes 226 and 228 may be fluidically connected to the inlet of the cell container 134 and may be used to extract a sample of the magnetically separated target cells after the magnetic separation process, or to inject culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, or a cell activation reagent into the cell container 134. The syringe 230 may be fluidically connected to the outlet of the cell container 134 and may be used to extract a sample of the genetically modified target cells after the transduction or transfection process is completed.

[0170] Continue to refer Figure 14 , the fluid circuit corresponding to system 500 includes sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, and acoustic separation device 302, all of which are interconnected by a fluid line network that passes through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separation devices 104, 105. The sample bags 102, 110, 112, 114A, 114B, 116, 220-224, syringes 226-230, cell container 134, acoustic separation device 302, and a network of fluid lines including conduits passing through magnetic separation devices 104 and 105 can be constructed, interconnected, sterilized, and provided as a disposable integrated kit. The electromechanical components of system 500 are located outside the tubing set and therefore do not come into contact with the fluid within the tubing set.

[0171] The first container or sample bag 102, which may contain target cells to be processed, is fluidly connected to the inlet of the conduit passing through the first magnetic separation device 104 by one or more fluid lines, which pass through the first pinch valve 148A, the first air detector 156A, the second air detector 158A, the peristaltic pump 160A and the occlusion sensor 162A, and is also fluidly connected to the inlet of the conduit passing through the second magnetic separation device 105 by one or more fluid lines, which pass through the first pinch valve 148B, the first air detector 156B, the second air detector 158B, the peristaltic pump 160B and the occlusion sensor 162B. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidly connected to the inlet of a conduit passing through the first magnetic separation device 104 by one or more fluid lines, which pass through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A, and is also fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines, which pass through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B, and an occlusion sensor 162B.

[0172] The inlet of cell container 134 is fluidically connected to the outlet of the conduit passing through first magnetic separation device 104 via one or more fluid lines, which pass through fourth pinch valve 154A, and is also fluidically connected to the outlet of the conduit passing through second magnetic separation device 105 via one or more fluid lines, which pass through fourth pinch valve 154B. Syringes 226 and 228 can be fluidically connected to the fluid lines between the inlet of cell container 134 and fourth pinch valves 154A and 154B. Sample bag 222, which can store waste fluid containing magnetically labeled non-target cells, is fluidically connected to the outlet of the conduit passing through first magnetic separation device 104 via one or more fluid lines, which pass through third pinch valve 152A, and is also fluidically connected to the outlet of the conduit passing through second magnetic separation device 105 via one or more fluid lines, which pass through third pinch valve 152B.

[0173] The outlet of the cell container 134 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., Figure 8A The side inlet 412 of the first container 116 is connected to the side inlet 412 of the first container 116. These fluid lines pass through the first peristaltic pump 312, the first occlusion sensor 316, and the first air detector 320. The syringe 230 can be fluidically connected to the fluid line connected to the outlet of the cell container 134. The third container or sample bag 116 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., Figure 8AThe sample bag 220 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., the central inlet 416 of the sample bag 220), which passes through the pinch valve 172, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324. Figure 8A ), these fluid lines pass through the pinch valve 174 , the second peristaltic pump 314 , the second occlusion sensor 318 and the first pinch valve 324 .

[0174] The fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic separation device 302 (eg, Figure 8A The sample bag 224, which can be used to receive waste fluid, is fluidically connected to the outlet of the acoustic separation device 302 (e.g., the central outlet 424 of the acoustic separation device 302) via one or more fluid lines. Figure 8A side outlet 420 ), these fluid lines pass through the second air detector 322 and the third pinch valve 328 .

[0175] The sample bag 114A of the culture component library 114 is fluidly connected to the inlet of the cell container 134 through one or more fluid lines, which pass through the clamping valve 168, the second clamping valve 150A, the second air detector 158A, the peristaltic pump 160A, the blockage sensor 162A, the first magnetic separation device 104 and the fourth clamping valve 154A, and through one or more fluid lines through the clamping valve 168, the second clamping valve 150B, the second air detector 158B, the peristaltic pump 160B, the blockage sensor 162B, the second magnetic separation device 105 and the fourth clamping valve 154B. Similarly, the sample bag 114B of the culture component library 114 is fluidly connected to the inlet of the cell container 134 via one or more fluid lines, which pass through the clamping valve 170, the second clamping valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104 and the fourth clamping valve 154A, and through one or more fluid lines through the clamping valve 170, the second clamping valve 150B, the second air detector 158B, the peristaltic pump 160B, the occlusion sensor 162B, the second magnetic separation device 105 and the fourth clamping valve 154B.

[0176] Embodiment 4 (Fourth embodiment): Positive magnetic separation, demagnetization bead labeling, acoustic purification

[0177] This embodiment uses a first magnetic separation device to separate or extract target cells with magnetic labels, a cell culture incubator genetically modifies the target cells with magnetic labels and removes the magnetic labels, especially when larger magnetic labels are used, and then an acoustic separation device is used to purify the genetically modified target cells.

[0178] Figure 15 6 is a block diagram illustrating an integrated system 600 and its components that can be used to produce CAR T cells. The integrated system 600 has control electronics, user interfaces, hardware, software, and firmware (not shown), and includes a first container or bag 102, a first magnetic separation device 104, a cell culture chamber 106, a second magnetic separation device 105, an acoustic separation device 302, a second container or bag 112 containing a buffer or solution for eluting target cells from the first magnetic separation device 104, a culture component reservoir 114, a third container or bag 116 containing a buffer or solution for operating the acoustic separation device 302, a fourth container or bag 110 for the final cell product, a fifth container or bag 602 containing a buffer or solution for eluting magnetic labels from the second magnetic separation device 105 and / or pre-treating the second magnetic separation device 105, and a plurality of fluidic lines providing fluidic interconnections between the various components of the integrated system 600 through fluidic lines 118-126, 306, 308, 604, 606.

[0179] A first container or bag 102 contains a fluid sample containing magnetically labeled target cells 132 (e.g., T cells). The fluid sample can include whole blood, leukapheresis products, PBMCs, and / or other leukapheresis products containing magnetically labeled target cells 132. The magnetic markers can attach to the target cells 132 during a culture process within the same first container or bag 102.

[0180] The first magnetic separation device 104 is connected to the first container or bag 102 via a fluid line 118 for extracting magnetically labeled target cells 132 from a fluid sample. Figure 3 、 4 The magnetic separation device 188 shown and described above has the same or substantially similar structure.

[0181] The second container or bag 112 is connected to the inlet of the first magnetic separation device 104 via a fluid line 120 and contains a buffer or solution for eluting the magnetically labeled target cells 132 remaining in the first magnetic separation device 104 after the fluid sample passes through. The buffer or solution in the second container or bag 112 can include a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0182] The cell culture chamber 106 can be used for cell genetic engineering, cell modification, cell transduction or cell transfection. The cell culture chamber 106 includes a cell container 134 therein, which is connected to the outlet of the first magnetic separation device 104 by a fluid line 122. The cell container 134 contains target cells 132 with magnetic labels, a buffer or solution for eluting cells from the first magnetic separation device 104, and optionally one or more buffers or solutions from a culture component library 114 connected to the cell container 134 by a fluid line 124. The cell culture chamber 106 can have one or more gas lines (e.g., CO2, not shown) connected thereto to provide an environment with a desired gas composition (e.g., 5.0% ± 0.1% CO2). The cell culture chamber 106 can also have a heating and cooling mechanism (not shown) that can maintain a constant temperature (e.g., 37.0 ± 0.1°C) indoors. In addition, the cell culture chamber 106 can further include an air circulation mechanism, such as a fan, to circulate air or other gas mixtures therein to make the temperature of the entire culture chamber 106 more uniform. The cell container 134 and its contents can be heated or cooled by convection from the surrounding environment in the cell culture chamber 106. The cell container 134 can also have porous or permeable portions, such as membranes and vents, that allow gas molecules from the surrounding environment to diffuse or flow, thereby exposing the contents of the cell container 134 to the air or other gas mixture within the cell culture chamber 106.

[0183] The culture component library 114 may include one or more of the following: culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection, reagents for removing magnetic labels from target cells during de-labeling.

[0184] The cell culture chamber 106 may further include devices capable of inducing opening of the target cell membrane, such as, but not limited to, cell electroporation, mechanical cell deformation, temperature, ultrasound, and optics, thereby allowing genetic material to enter the target cell from the surrounding culture medium in the cell container 134. Genetic material can also be introduced into the target cell by droplet-based genetic material injection or by permeation of the target cell membrane.

[0185] The cell culture chamber 106 can provide an environment for the transduction process. For example, a lentivirus or other virus can be used to transfer genetic material into target cells to induce the growth of chimeric antigen receptors (CARs) on the cell surface. The magnetic marker attached to the target cell 132 can also be removed from the target cell by adding a de-labeling agent from the culture component library 114 to the cell container 134 before, during, or after the genetic modification process (e.g., transduction or transfection).

[0186] The second magnetic separation device 105 is connected to the cell container 134 in the cell culture chamber 106 via a fluid line 126 and is used to remove free magnetic labels that are stripped from the genetically modified target cells in the solution and / or culture medium mixture in the cell container 134. The mixture may include a buffer for eluting the genetically modified target cells from the first magnetic separation device 104, one or more reagents for transduction or transfection, and a reagent for removing the labels of the target cells 132. The second magnetic separation device 105 retains the free magnetic labels when the solution and / or culture medium mixture (containing the genetically modified target cells from which the labels have been removed) flows through the second magnetic separation device 105 and enters the inlet of the acoustic separation device 302 through the fluid line 604. The second magnetic separation device 105 can have a structure that is the same as or substantially similar to that of the magnetic separation device 188, and its cross-sectional view is shown in FIG. Figure 3 、 Figure 4 shown and described above.

[0187] In an alternative embodiment, the labeled target cells can flow from the second magnetic separation device 105 to a container or bag, and then further to the inlet of the acoustic separation device 302. The container or bag can serve as a reservoir, temporarily storing the fluid therein, so as to allow the second magnetic separation device 105 downstream thereof and the acoustic separation device 302 upstream thereof to operate at different flow rates as needed.

[0188] The fifth container or bag 602 is connected to the inlet of the second magnetic separation device 105 via a fluid line 606 and contains a buffer or solution for eluting free magnetic labels remaining in the second magnetic separation device 105 after the fluid sample passes through. The buffer or solution in the fifth container or bag 602 can include a PBS-based solution, a culture medium-based solution, human serum, glucose, or any combination thereof.

[0189] The acoustic separation device 302 is connected to the outlet of the second magnetic separation device 105 via a fluid line 604, and is used to extract the genetically modified target cells from the solution and / or culture medium mixture output from the second magnetic separation device 105. The acoustic separation device 302 may have a Figures 8A-8E The acoustic separation device 428 shown has the same or substantially similar structure.

[0190] The third container or bag 116 is connected to the inlet of the acoustic separation device 302 via a fluid line 306 and contains a buffer or solution that may be used as a sheath fluid when acoustically separating the genetically modified target cells in the acoustic separation device 302. The buffer or solution may also be used as a storage solution for preserving the genetically modified target cells in the fourth container or bag 110. The buffer or solution in the third container or bag 116 may include a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof.

[0191] A fourth container or bag 110 is connected to the outlet of the acoustic separation device 302 via a fluid line 308 and contains genetically modified target cells in a buffer or solution that can be administered intravenously to a patient.

[0192] The network of fluid lines 118-126, 306, 308, 604, 606, the containers or bags 102, 110, 112, 116, 602, the cell container 134, and the fluid lines passing through the first magnetic separation device 104 and the second magnetic separation device 105 and the acoustic separation device 302 can be constructed, interconnected and provided as a disposable integrated tubing set, which can be sterile and sealed from the surrounding environment.

[0193] Continue to refer Figure 15 The process begins by providing a first container or bag 102 containing a first fluid sample with magnetically labeled target cells 132. For a previously frozen sample, the first fluid sample can be prepared by first thawing the frozen sample and then extracting the target cells and other biological objects (if any) from the thawed sample fluid by centrifugation. The resulting target cells and other biological objects (if any) are resuspended in a buffer and filtered using a mesh size of 15 to 100 microns. A reagent containing a magnetic label is added to the filtered buffer containing the target cells to form the first fluid sample. Alternatively, the target cells can be magnetically labeled by an indirect process by first adding a reagent containing an intermediate link that attaches to the target cells and then adding a reagent containing a magnetic label attached to the intermediate link.

[0194] The first fluid sample flows through fluid line 118 into the inlet of the magnetic separation device 104 for magnetic separation. As the first fluid sample flows through the magnetic separation device 104, the magnetically labeled target cells 132 are retained in the magnetic separation device 104 by the magnetic field, while the depleted first fluid sample exits the magnetic separation device 104 and enters a waste container or bag (not shown). After the first fluid sample has completely passed through the magnetic separation device 104, the magnetic field acting on the magnetically labeled target cells 132 is weakened or removed, and the first eluent, such as a buffer, in the second container or bag 112 flows through fluid line 120 into the magnetic separation device 104, eluting the magnetically labeled target cells 132 and entering the cell container 134 through fluid line 122. In addition, one or more solutions from the culture component library 114, such as, but not limited to, culture medium, vectors for cell transduction or transfection, lentivirus for transduction, or buffers or solutions for non-viral transfection, are injected into the cell container 134 through fluid line 124 to modify the genetic structure of the magnetically labeled target cells 132. Target cells 132 (e.g., T cells) with magnetic labels can be transduced to express CAR by contacting with a vector carrying a CAR construct, including viral vectors such as lentiviruses and retroviruses, and non-viral vectors such as plasmids, lipid nanoparticles, and mRNA.

[0195] The transduction or transfection process is carried out in the cell culture chamber 106, where the target cells 132 with magnetic labels are immersed in a transduction or transfection medium comprising a first eluent and one or more solutions from the culture component library 114. During the transduction or transfection process, the temperature and gas composition within the cell culture chamber 106 can be controlled. For example, when the genetic structure of the target cells 132 with magnetic labels is modified in the cell container 134, the temperature and gas composition within the cell culture chamber 106 can be maintained at 37.0±0.1°C and 5.0%±0.1% CO2, respectively, for a duration ranging from 10 minutes to 72 hours. After the transduction or transfection process is completed, a reagent can be injected into the cell container 134 from the culture component library 114 to remove the magnetic labels on the target cells. Alternatively, the de-labeling process can be performed before or during the transduction or transfection process.

[0196] After the genetic modification and delabeling process of the target cells is completed, a buffer for facilitating the subsequent acoustic sorting process can be added from the culture component library 114 to the transduction or transfection medium and delabeling reagent in the cell container 134. The mixture of the transduction or transfection medium, the delabeling reagent, and the buffer for facilitating the acoustic sorting process (if any) forms a second fluid sample that does not contain magnetically labeled, genetically modified target cells.

[0197] The second fluid sample then flows into the inlet of the second magnetic separation device 105 through fluid line 126 to extract the free magnetic markers through a second magnetic separation process. When the second fluid sample flows through the second magnetic separation device 105, the free magnetic markers removed from the genetically modified target cells are retained in the second magnetic separation device 105 by the magnetic field, while the genetically modified target cells, carried by the depleted second fluid sample (i.e., without the free magnetic markers), leave the second magnetic separation device 105 through fluid line 604 and enter the acoustic separation device 302.

[0198] by Figures 8A-8E In the illustrated acoustic separation device 428, for example, a depleted second fluid sample containing genetically modified target cells 432 is introduced into the side inlet 412 via fluid line 604, while a buffer solution is introduced into the central inlet 416 from the third container or bag 116 via fluid line 306. The depleted second fluid sample containing the genetically modified target cells 432 and non-target biological objects / debris 434 (if any) is introduced into the separation channel 410 near the upstream end of the separation channel 410 via a pair of side inlet channels 414 as two laminar flows 436 and 438 flowing close to the side walls. The two laminar flows 436 and 438 in the separation channel 410 are separated by a central flow 440 of buffer solution from the central inlet 416. The central flow 440 of buffer solution can exhibit laminar behavior and act as a sheath flow, slowing or preventing the migration of non-target biological objects / debris 434 toward the pressure node located at the center of the separation channel 410. As the depleted second fluid sample proceeds downstream along the separation channel 410, acoustic radiation pressure pushes the genetically modified target cells 432 into the central stream 440 and toward the pressure node located at the center of the separation channel 410, while the non-target biological objects / debris 434 remain mostly within the two laminar flows 436 and 438 near the side walls. At the downstream end of the separation channel 410, the genetically modified target cells 432, carried by the buffer, exit the acoustic separation device 428 through the central outlet 424 and enter the fourth container or bag 110 through the fluid line 308. The further depleted second fluid sample, including the non-target biological objects / debris 434, flows as laminar flows 436 and 438 near the side walls and is diverted to the side outlet 420 through a pair of side outlet channels 422.

[0199] After the acoustic sorting process is complete, the fourth container or bag 110 containing the genetically modified target cells can be separated from the rest of the integrated system 600 without exposing its contents to the atmosphere. The contents of the fourth container or bag 110 can be administered intravenously to a patient.

[0200] Figure 9FIG6 is a front view of an integrated system 600 that can be used to produce CAR T cells from whole blood or T cell-containing samples according to the above process. System 600 can utilize closed fluidic components to form a closed processing system that can be deployed in facilities that lack a strict sterile or clean environment, such as treatment facilities, thereby alleviating many common logistical issues encountered in autologous treatments.

[0201] Figure 9 An automated production system 600 is shown, comprising a housing or enclosure structure 140, first and second magnetic separation modules 142A, 142B and acoustic separation module 310 residing within the enclosure structure 140, an incubation module 144, a sample rack 146 mounted on top of the enclosure structure 140, and first and second sample bag racks 164, 166. Fluid lines and sample bags connected thereto, which may be provided as integrated disposable tubing sets, and a computer for controlling the system 600 are omitted from the figure to provide an unobstructed view of the magnetic separation modules 142A, 142B and the acoustic separation module 310. Production system 600 is similar to systems 300 and 500, except that the routing of the fluid lines is different, as described below.

[0202] The culture module 144 includes a culture chamber 106 located on top of a rocking base 147. Each magnetic separation module 142A, 142B includes a first pinch valve 148A, 148B, a second pinch valve 150A, 150B, a third pinch valve 152A, 152B, a fourth pinch valve 154A, 154B, a first air detector 156A, 156B, a second air detector 158A, 158B, a peristaltic pump 160A, 160B, an occlusion sensor 162A, 162B, and magnetic separation devices 104, 105. The acoustic separation module 310 includes a first peristaltic pump 312, a second peristaltic pump 314, a first occlusion sensor 316, a second occlusion sensor 318, a first air detector 320, a second air detector 322, an acoustic separation device 302, and first, second, and third pinch valves 324-328.

[0203] The system 600 may further include additional pinch valves 168, 170, 608 located on one side of the housing structure 140, and additional pinch valves 172, 174 located on the other side of the housing structure 140. The electromechanical assemblies 104-106, 147, 148A / B-162A / B, 168-174, 312-328, 608 may be controlled or automated by a computer or microprocessor (not shown).

[0204] Each magnetic separation device 104, 105 may have Figure 3 and 4The same or substantially similar structure as the magnetic separation device 188 shown and described above. Other magnetic flux sources and pillarless magnetic separation devices, such as those disclosed in U.S. application Ser. No. 18 / 072,362 (incorporated herein by reference), can also be used to separate magnetically labeled target cells from a fluid sample.

[0205] The acoustic separation device 302 may have Figures 8A-8E The acoustic separation device 428 shown and described above may have the same or substantially similar structure.

[0206] The culture module 144 includes a cell culture chamber 106 atop a rocking base 147. The culture chamber 106 is equipped with a heater to maintain a uniform temperature between room temperature and 50°C. The CO2 concentration within the culture chamber 106 can be varied between 0% and 10% to provide an optimal environment for the cells. The rocking base 147 can move the culture chamber 106 at a speed of 2 to 4 revolutions per minute. The rocking motion of the culture chamber 106 caused by the rocking base 147 promotes uniform mixing of the fluid contents within the cell container 134. During cell activation, transduction, or expansion, the rocking base 147 can remain stationary. After the culture process, the rocking base 147 can rock the cell container 134 within the culture chamber 106, flushing the fluid within the cell container 134 from the surface of the container. The rocking base 147 can also be used to facilitate drainage of the cell container 134 by tilting the culture chamber 106 into a fixed position, allowing the end of the outlet tube to reach the bottom of the fluid within the cell container 134.

[0207] The system 600 has a modular design and includes two magnetic separation modules 142A, 142B, one acoustic separation module 310, and one incubation module 144. The modules 142A, 142B, 144, and 310 can be fluidically connected in series, in parallel, or a combination thereof through a closed fluidic assembly that includes sample bags, cell containers, an acoustic separation device, and all fluid lines (including a conduit passing through the magnetic separation device).

[0208] Figure 16 is a perspective view of a system 600 for manufacturing CAR T cells using a closed fluidic assembly in the form of an integrated tubing set. For clarity, Figure 9 The electromechanical components labeled in FIG are not relabeled in this figure to clearly illustrate the path of the fluid circuit. The perspective view also shows the pinch valves 152B, 154A, 154B, 328, which were previously obscured by the cell culture chamber 106, and Figure 9Pinch valves 172, 174 are shown on one side of the housing structure 140 in a front view. The lid and a portion of the sidewall of the culture chamber 106 are removed to expose the cell container 134, which is in the form of a conventional cell culture flask, within the culture chamber 106. The lid of the cell culture flask 134 includes an air filter that vents air into the environment within the culture chamber 106 while maintaining the sterility of the flask 134. The second bag rack 166 is also omitted from the figure to illustrate the sample bags 110, 224 within the rack 166. Figure 17 corresponds to Figure 16 A fluid circuit diagram of system 600 is shown.

[0209] refer to Figure 9 、 Figure 16 、 Figure 17 Unlike the fluidic components of systems 100, 300, and 500, the fluidic component of system 600 is not configured to simultaneously extract magnetically labeled target cells from the first fluid sample using first magnetic separation device 104 and second magnetic separation device 105 for magnetic separation. First magnetic separation device 104 is used to extract magnetically labeled target cells from the first fluid sample, while second magnetic separation device 105 is used to remove free magnetic labels after the magnetic labels are removed from the target cells. Acoustic separation device 302 is then used to purify non-magnetic target cells.

[0210] The first, second, third and fourth containers or bags 102, 112, 116, 110 are in the form of sample bags. Sample bags 102 and 112 are both fluidically connected to the inlet of the first magnetic separation device 104. In addition to sample bag 116, another sample bag 220 (which may also contain a cryopreservation solution, a storage medium, a PBS-based solution, a saline-based solution, human serum, glucose, or any combination thereof) is also fluidically connected to the inlet of the acoustic separation device 302. The culture component library 114 includes at least three sample bags 114A-114C, which are fluidically connected to the cell container 134 via the magnetic separation device 104. Each sample bag 114A, 114B, 114C may include culture medium, a vector for cell transduction or transfection, a lentivirus for transduction, a buffer or solution for non-viral transfection, a cell activation reagent, or a reagent for stripping a magnetic label from the target cells (i.e., a de-labeling reagent). Prior to the transduction or transfection process, the target T cells in the cell container 134 may be activated by flowing a cell activation reagent (eg, a solution containing CD3 / CD28 magnetic beads) from one of the sample bags 114A- 114C into the cell container 134 .

[0211] The fluidics assembly of system 600 further includes a sample bag 222 fluidically connected to the outlets of the first and second magnetic separation devices 104, 105 for collecting waste from the devices 104, 105, and another sample bag 224 fluidically connected to the outlet of the acoustic separation device 302 for collecting waste from the device 302. The sample bags 222 for collecting waste from the first and second magnetic separation devices 104, 105 can be placed in a first bag rack 164. Sample bags 110, 224 for collecting the final cell product and waste from the acoustic separation device 302, respectively, can be placed in a second bag rack 166. All other sample bags 102, 112, 114A-114C, 116, 220 that provide materials for the manufacturing process can be hung on the sample rack 146. The fluidics assembly of system 600 can include additional sample bags for various purposes. Each sample bag 102, 110, 112, 114A-114C, 116, 220-224 can have an inlet port with a Luer connector connected thereto and an outlet port with a drip chamber. In addition, the flow of the inlet and outlet ports of each sample bag can be controlled or regulated by one or more manually tightened clamps attached thereto.

[0212] The fluidic components of system 600 may further include syringes 226, 228, and 230. Syringes 226 and 228 can be fluidically connected to the inlet of cell container 134 and can be used to extract a sample of magnetically separated target cells after the magnetic separation process, or to inject culture medium, vectors for cell transduction or transfection, lentivirus for transduction, buffers or solutions for non-viral transfection, cell activation reagents, or delabeling reagents into cell container 134. Syringe 230 can be fluidically connected to the outlet of cell container 134 or the outlet of the second magnetic separation device 105 and can be used to extract a sample of genetically modified target cells after the transduction or transfection process is completed.

[0213] Continue to refer Figure 17, the fluid circuit corresponding to system 600 includes sample bags 102, 110, 112, 114A-114C, 116, 220-224, syringes 226-230, cell container 134 and acoustic separation device 302, all of which are interconnected by a fluid line network that passes through electromechanical components such as pinch valves 148A / B-154A / B, 168-172, 324-328, air detectors 156A / B, 158A / B, 320, 322, occlusion sensors 162A, 162B, 316, 318, peristaltic pumps 160A, 160B, 312, 314, and magnetic separation devices 104, 105. The sample bags 102, 110, 112, 114A-114C, 116, 220-224, syringes 226-230, cell container 134, acoustic separation device 302, and a network of fluid lines including conduits passing through magnetic separation devices 104 and 105 can be constructed, interconnected, sterilized, and provided as a disposable integrated kit. The electromechanical components of system 600 are located outside the tubing set and therefore do not come into contact with the fluid within the tubing set.

[0214] A first container or sample bag 102, which may contain target cells to be processed, is fluidically connected to the inlet of a conduit passing through the first magnetic separation device 104 via one or more fluid lines, which pass through a first pinch valve 148A, a first air detector 156A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A. A second container or sample bag 112, which may contain a buffer solution or culture medium, is fluidically connected to the inlet of a conduit passing through the first magnetic separation device 104 via one or more fluid lines, which pass through a second pinch valve 150A, a second air detector 158A, a peristaltic pump 160A, and an occlusion sensor 162A, and is also fluidically connected to the inlet of a conduit passing through the second magnetic separation device 105 via one or more fluid lines, which pass through a second pinch valve 150B, a second air detector 158B, a peristaltic pump 160B, and an occlusion sensor 162B. In the arrangement shown, the first magnetic separation device 104 and the second magnetic separation device 105 use the same buffer or culture medium solution stored in the second container or sample bag 112, rather than Figure 15 A separate container or sample bag 112, 602 is shown. However, the separate container or sample bag 602 may be fluidly connected to an inlet of a conduit passing through the second magnetic separation device 105 for eluting free magnetic labels collected by the device 105.

[0215] The inlet of the cell container 134 is fluidically connected to the outlet of the conduit passing through the first magnetic separation device 104 via one or more fluid lines, which pass through a third pinch valve 152A. Syringes 226 and 228 can be fluidically connected to the fluid line between the inlet of the cell container 134 and the third pinch valve 152A. The sample bag 222, which can store waste fluid containing non-target cells and / or free magnetic markers, is fluidically connected to the outlet of the conduit passing through the first magnetic separation device 104 via one or more fluid lines, which pass through a fourth pinch valve 154A, and is also fluidically connected to the outlet of the conduit passing through the second magnetic separation device 105 via one or more fluid lines, which pass through a fourth pinch valve 154B.

[0216] The outlet of the cell container 134 is fluidly connected to the inlet of a conduit passing through the second magnetic separation device 105 by one or more fluid lines, which pass through a first pinch valve 148B, a first air detector 156B, a second air detector 158B, a peristaltic pump 160B and an occlusion sensor 162B.

[0217] The outlet of the conduit passing through the second magnetic separation device 105 is fluidly connected to the inlet of the acoustic separation device 302 (eg, Figure 8A The side inlet 412 of the second magnetic separation device 105 is connected to the side inlet 412 of the second magnetic separation device 105. These fluid lines pass through the third pinch valve 152B, the first peristaltic pump 312, the first occlusion sensor 316, and the first air detector 320. The syringe 230 can be fluidically connected to the fluid line connected to the outlet of the cell container 134, or as shown, to the outlet of the conduit passing through the second magnetic separation device 105. The third container or sample bag 116 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., Figure 8A The sample bag 220 is fluidically connected to the inlet of the acoustic separation device 302 (e.g., the central inlet 416 of the sample bag 220), which passes through the pinch valve 172, the second peristaltic pump 314, the second occlusion sensor 318, and the first pinch valve 324. Figure 8A ), these fluid lines pass through the pinch valve 174 , the second peristaltic pump 314 , the second occlusion sensor 318 and the first pinch valve 324 .

[0218] The fourth container or sample bag 110 is fluidly connected to the outlet of the acoustic separation device 302 (eg, Figure 8A The sample bag 224, which can be used to receive waste fluid, is fluidically connected to the outlet of the acoustic separation device 302 (e.g., the central outlet 424 of the acoustic separation device 302) via one or more fluid lines. Figure 8A side outlet 420 ), these fluid lines pass through the second air detector 322 and the third pinch valve 328 .

[0219] The sample bag 114A of the culture component library 114 is fluidically connected to the inlet of the cell container 134 via one or more fluid lines, which pass through the pinch valve 168, the second pinch valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104, and the third pinch valve 152A. The sample bag 114B of the culture component library 114 is fluidically connected to the inlet of the cell container 134 via one or more fluid lines, which pass through the pinch valve 170, the second pinch valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104, and the third pinch valve 152A. Similarly, the sample bag 114C of the culture component library 114 is fluidly connected to the inlet of the cell container 134 via one or more fluid lines, which pass through the clamping valve 608, the second clamping valve 150A, the second air detector 158A, the peristaltic pump 160A, the occlusion sensor 162A, the first magnetic separation device 104 and the third clamping valve 152A.

[0220] As will be appreciated by those skilled in the art, the processes described in Embodiments 1 to 4 can be practiced using other systems or instruments not described herein. For example, the initial T cell magnetic separation / extraction and / or final magnetic collection / purification can be performed using a column-based magnetic separation device; or the magnetic separation device, acoustic separation device, or cell container may not be fluidically connected or integrated into the system (i.e., a discrete instrument).

[0221] Example

[0222] The following examples are intended to illustrate the present invention but not to limit it.

[0223] The target cell recovery reported in this article was calculated by dividing the number of target cell events or cells in the fluid sample after magnetic or acoustic sorting by the number of target cell events or cells in the initial fluid sample before sorting, as measured by flow cytometry (CytoFlex, Beckman Coulter). The target cell purity reported in this article was calculated by dividing the number of target cell events or cells by the total number of all events or cells in the fluid sample, as measured by flow cytometry.

[0224] Example 1: Production of CAR T cells from fresh leukapheresis products using positive magnetic separation and positive magnetic purification

[0225] This embodiment uses equipment such as Figure 2 、 Figure 5 、 Figure 6The system 100 of closed fluidic components shown and described above includes three magnetic modules 142A-142C and a culture module 144. The closed fluidic components include sample bags 102, 110, 112, 114A, 114B, 116, 220-224, a cell container 134 in the form of a cell culture flask, conduits passing through magnetic separation devices 104, 105, 108, and fluid lines connecting these components.

[0226] The sample preparation process was to incubate the leukocyte separation product sample (about 1 / 10 of the leukocyte separation product, containing about 6x10^8 cells) with CD4 / CD8 magnetic beads (Beijing T&L Biotechnology Co., Ltd.) at room temperature for 30 minutes while shaking on a mixer to magnetically label T cells. After incubation, the leukocyte separation product sample was washed with buffer ( MAG Buffer, Applied Cells) was diluted to a cell concentration of approximately 3 × 10 7 cells / ml, with a total volume of about 20 ml, to obtain a first fluid sample, which is then transferred to the first sample bag 102. It is noteworthy that, as reported in U.S. patent application Ser. No. 18 / 795,047 (incorporated herein by reference in its entirety), a method can also be used. Figure 3 and 4 The magnetic separation device shown extracts or separates T cells directly from whole blood without the need for leukapheresis or buffy coat extraction.

[0227] The process of separating or extracting T cells from a first fluid sample begins by flowing fluid from a first sample bag 102 through a pair of magnetic separation devices 104 and 105 at a total flow rate of 1 ml / min. As the first fluid sample flows through the magnetic separation devices 104 and 105, the magnetically labeled T cells are retained within the devices 104 and 105 by the magnetic field, while the depleted first fluid sample exits the devices 104 and 105 and is collected by a sample bag 222. After the first fluid sample has completely passed through the magnetic separation devices 104 and 105, the magnetic field acting on the magnetically labeled T cells is removed, and a first eluent, comprising T cell culture medium (OptiVitro, ExcellBio) containing IL-2 (300 IU / ml), IL-7 (10 ng / ml), and IL-15 (10 ng / ml) (TL), flows from a second sample bag 112 into the magnetic separation devices 104 and 105, eluting the magnetically labeled T cells into a cell culture flask 134 located within a cell culture chamber 106.

[0228] Figure 18ADot plots are shown for a first fluid sample (including an initial leukapheresis product sample before the magnetic separation process) and a culture medium containing T cells extracted from the magnetic separation process. Flow cytometry data indicate that the magnetic separation process increased the purity of the T cells (as a percentage of the total white blood cell count) from 60% to 96%, and that 59% of the T cells were recovered from the magnetic separation process (i.e., the recovery rate). Figure 18B The flow cytometry data further showed that the CD4+ / CD8+ ratio of T cells remained relatively constant after the magnetic separation process.

[0229] After the magnetic separation process, the process of activating the T cells in the cell culture flask 134 begins by flowing the activation reagent (including ActCel CD3 / CD28 magnetic beads, Beijing T&L Biotechnology Co., Ltd.) diluted in T cell culture medium from the sample bag 114A into the cell culture flask 134. After the activation reagent is added to the cell culture flask 134, the cell culture chamber 106 is rocked on the rocking base 147 to evenly mix the fluid mixture in the cell culture flask 134. The T cells are then incubated with the CD3 / CD28 magnetic beads in the cell culture chamber 106 at 37°C and 5% CO2 for 24 hours.

[0230] Figure 19 The figure shows the expression of activation markers CD25 and CD69 on T cells after 24 hours of incubation with CD3 / CD28 magnetic beads. The figure shows that 80%, 74% and 69% of T cells express CD25, CD69 and CD25+CD69 (double positive) activation markers, respectively.

[0231] After the activation process, the process of transducing the T cells in the cell culture flask 134 begins by flowing a lentiviral solution (including CD19 CAR-T lentivirus suspended in T cell culture medium, Jiangsu Hill Gene Biopharmaceutical Co., Ltd.) from the sample bag 114B into the cell culture flask 134 at a multiplicity of infection (MOI) of 5. After the lentiviral solution is added to the cell culture flask 134, the cell culture chamber 106 is rocked on the rocking base 147 to evenly mix the fluid mixture in the cell culture flask 134. The T cells are then incubated with the lentivirus in a 37°C, 5% CO2 environment in the cell culture chamber 106 for 24 hours.

[0232] After the transduction process, the T cell collection / purification process begins by flowing the second fluid sample (including T cells and residual lentivirus) in the cell culture bottle 134 through the magnetic separation device 108 at a flow rate of 0.5 ml / min. As the second fluid sample flows through the magnetic separation device 108, the magnetically labeled T cells are retained in the device 108 by the magnetic field, while the depleted second fluid sample containing residual lentivirus leaves the device 108 and is collected by the sample bag 224. After the second fluid sample has completely passed through the magnetic separation device 108, the magnetic field acting on the magnetically labeled T cells is removed, and the second eluent, including the buffer ( MAG Buffer (Applied Cells) flows from the third sample bag 116 into the magnetic separation device 108, eluting the T cells into the fourth sample bag 110 for subsequent analysis, including flow cytometry. The magnetic collection / purification process recovered 91% of the T cells from the second fluid sample after activation and transduction. Alternatively, the cryopreservation solution stored in the sample bag 220 can be used as the second eluent to preserve the T cells in the fourth sample bag 110 for cryopreservation.

[0233] Figure 20A and Figure 20B are dot plots corresponding to the final T cell product after the transduction process and magnetic collection / purification process. Figure 20A It was shown that 65% of T cells expressed CAR. In addition, Figure 20B It was shown that 88% of CAR T cells expressed the CCR7 marker (memory T cells), and more specifically, 54% of CAR T cells were T stem cell memory (TSCM) cells. The percentage of TSCM cells produced by the current method is significantly higher than that of conventional production methods that require T cell expansion.

[0234] Although the present invention has been shown and described in terms of certain preferred embodiments, those skilled in the art will undoubtedly devise certain changes and modifications that still encompass the true spirit and scope of the present invention. Accordingly, the scope of the present invention should be determined by the appended claims and their legal equivalents, rather than by the examples given.

[0235] Any element in a claim that does not explicitly state “means for” performing a specified function or “step for” performing a specified function should not be construed as constituting a violation of 35 U.S.C. 112, In particular, the use of "step" in the claims herein is not intended to invoke 35 U.S.C. 112, regulations.

Claims

1. A method for producing a T cell population expressing a chimeric antigen receptor (CAR), comprising the following steps: providing a fluid sample containing a T cell population; labeling the T cell population with magnetic beads; extracting a population of T cells from the fluid sample by passing the fluid sample through a magnetic separation device; transforming the T cell population to express a chimeric antigen receptor (CAR) by contacting the T cell population with a vector carrying a CAR construct; as well as The T cell population is collected by passing the solution through the magnetic separation device or another magnetic separation device.

2. The method according to claim 1, further comprising the step of activating the T cell population before transforming the T cell population.

3. The method according to claim 2, wherein the step of activating the T cell population is performed by contacting the T cell population with CD3 / CD28 magnetic beads. The method of claim 1 , wherein the vector is a lentivirus.

5. The method of claim 1, wherein the vector is a retrovirus. The method of claim 1 , wherein the fluid sample comprises whole blood.

7. The method of claim 1, wherein the fluid sample comprises a leukapheresis product.

8. A method for producing a T cell population expressing a chimeric antigen receptor (CAR), comprising the following steps: providing a fluid sample containing a T cell population; labeling the T cell population with magnetic beads; extracting the T cell population from the fluid sample by passing the fluid sample through a first conduit passing through a first magnetic separation device; transforming the T cell population to express a chimeric antigen receptor (CAR) by contacting the T cell population with a lentivirus population contained in a solution in a culture vessel; and collecting the T cell population by passing the solution through a second conduit passing through a second magnetic separation device, The first conduit, the culture container and the second conduit are fluidically connected via a fluid line network.

9. The method of claim 8, wherein the first conduit, the culture container, the second conduit, and the fluid line network are components of a closed fluid system.

10. The method according to claim 8, further comprising the step of activating the T cell population before transforming the T cell population.

11. The method of claim 8, wherein the fluid sample comprises whole blood.

12. The method of claim 8, wherein the first magnetic separation device and the second magnetic separation device each comprise: a permanent magnet having a first pole and a second pole; a central flux guide comprising a central tip having a conical shape and a central base magnetically coupled to the first pole; a bottom flux guide magnetically coupled to the second pole; a first side flux guide comprising a first side tip and a first side base magnetically coupled to the second pole through the bottom flux guide; a second side flux guide comprising a second side tip and a second side base magnetically coupled to the second pole via a bottom flux guide; The first side magnetic flux guide and the second side magnetic flux guide are located on opposite sides of the central magnetic flux guide, and the first side tip and the second side tip are bent toward the central tip. 13 . The method of claim 12 , wherein the first magnetic separation device and the second magnetic separation device each further comprise a floating magnetic flux guide that pushes the first conduit or the second conduit toward the central tip and the first and second side tips.

14. A method for producing a population of T cells expressing a chimeric antigen receptor (CAR), comprising the steps of: providing a fluid sample containing a T cell population; Label T cell populations with magnetic beads; extracting a population of T cells from a fluid sample by passing the fluid sample through a conduit passing through a magnetic separation device; transforming the T cell population to express a chimeric antigen receptor (CAR) by contacting the T cell population with a lentivirus population contained in a solution in a culture vessel; The T cell population is collected by passing the solution through an acoustic separation device, The conduit, the culture container and the acoustic separation device are fluidically connected via a fluid line network.

15. The method according to claim 14, further comprising the step of activating the T cell population before transforming the T cell population.

16. The method of claim 14, wherein the conduit, the culture container, the acoustic separation device, and the fluid line network are components of a closed fluid system.

17. The method of claim 14, wherein the acoustic separation device comprises: a planar substrate having a first planar surface and a second planar surface; a network of channels recessed from said first planar surface; a cover attached to the first planar surface and covering the channel network; as well as One or more piezoelectric transducers are attached to an exterior surface of the cover, opposite an interior surface of the cover facing the channel network.

18. The method of claim 14, wherein the magnetic separation device comprises: a permanent magnet having a first pole and a second pole; a central flux guide comprising a central tip having a conical shape and a central base magnetically coupled to the first pole; a bottom flux guide magnetically coupled to the second pole; a first side flux guide comprising a first side tip and a first side base magnetically coupled to a second pole via a bottom flux guide; a second side flux guide including a second side tip and a second side base magnetically coupled to the second pole via a bottom flux guide; The first side magnetic flux guide and the second side magnetic flux guide are located on opposite sides of the central magnetic flux guide, and the first side tip and the second side tip are bent toward the central tip.

19. The method of claim 18, wherein the magnetic separation device further comprises a floating magnetic flux guide that urges the catheter toward the central tip and the first and second side tips.

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