Integrated bioprocess container for activation and expansion of t cells

Abstract

A bioprocessing container for engineering, activating, and expanding T cells, and a method for using such a bioprocessing container, are provided. The bioprocessing container includes a container body, a retractable impeller assembly, and an adjustment knob structure. The configuration of the bioprocessing container solves the problem of T cell activation in a rotary flask, reducing the time, cost, and contamination risks associated with current rotary flask technology.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 600,969, filed November 20, 2023, pursuant to 35 USC §119, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to the field of bioprocessing, and more specifically, to a bioprocessing container for generating T cells, including engineered T-cell immunotherapies derived from patient-derived donor cells such as chimeric antigen receptor-modified T cells and other engineered T-cell receptor therapy cells. Background Technology

[0004] Bioprocessing is a broad term used to describe the upstream and downstream processes associated with the production of therapeutic products of interest from cultured cells. One area of ​​interest is engineered cell immunotherapy, which uses a patient's own T cells to create a tailored therapy. Specifically, chimeric antigen receptor-modified T cell (CAR-T cell) and engineered T cell receptor (TCR-T cell) immunotherapies have shown significant success in treating some hematologic malignancies and have the potential to treat other cancer types. Producing engineered cell therapies is complex and extremely expensive, resulting in single treatments costing patients between $350,000 and $450,000. Engineered cell therapies begin by extracting and purifying the patient's blood to obtain T cells with certain markers. The extracted T cells are then engineered to produce certain proteins on the outside of the T cells in the form of membrane receptors, which help to recognize and assist in the destruction of targeted malignant cells. In CAR-T cell therapies targeting certain types of blood cancers, the chimeric antigen receptor (CAR) gene is incorporated into activated T cells. In TCR-T cell therapy targeting certain types of solid tumors, specific cancer antigen-specific T-cell receptors (TCRs) can be incorporated into activated T cells. Once engineered T cells (CAR-T cells or TCR-T cells) are created, they are then activated by exposing them to certain types of antibodies conjugated to colloidal polymer nanomatrix or paramagnetic beads. These engineered T cells must proliferate to therapeutic levels; this stage is called expansion. Finally, these proliferated CAR-T cells are administered back to the patient as a therapeutic agent.

[0005] Platforms exist for preparing these cells, ranging from highly manual to fully automated. However, current platforms for engineered cell therapies are open systems, meaning they require handling and multiple transfer steps throughout the process. The cell transfer and handling in current platforms significantly increases the risk of contamination per batch and makes the preparation process difficult to automate. This is a major drawback of existing platforms, as these shortcomings increase the complexity and cost of engineered cell therapies.

[0006] Highly automated, closed-loop integrated systems offer minimal manipulation but come with significant equipment costs, which are typically associated with such devices. There is a need for closed-loop systems to assist in the research and development of engineered cell therapies that are both economical and minimize the processing and multiple transfer steps that currently plague systems. This disclosure provides an economical closed-loop system solution that minimizes processing and transfer steps. Summary of the Invention

[0007] According to some aspects of this disclosure, a bioprocessing apparatus is provided, comprising a container body, one or more constricted inlet ports, an impeller assembly, a rod, and an adjustment knob structure. The container body includes a top portion and a bottom portion. The rod includes a first end and a second end. The impeller assembly includes a shaft and a plurality of planar blades extending from the shaft. The shaft of the impeller assembly is connected to the first end of the rod, and the rod extends through the adjustment knob structure. The second end of the rod is connected to a screw external to the adjustment knob structure. Additionally, the impeller assembly is partially retractable. This bioprocessing container is configured for activating and expanding T cells.

[0008] In some embodiments, the second end of the rod includes a series of helical grooves that connect to an internal helical groove on the screw. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body. In some embodiments, the retracted impeller assembly remains retracted when the screw is loosened.

[0009] In some embodiments, the impeller assembly includes a magnet. In some embodiments, when the impeller assembly is in its maximum retracted state, it does not experience magnetic interference with the magnetic bead.

[0010] In some embodiments, a first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly. In some embodiments, the impeller assembly is rotatable against the rod.

[0011] According to some aspects of this disclosure, a bioprocessing container is provided, the bioprocessing container comprising: a container body including a top portion and a bottom portion; one or more necked inlet ports; an impeller assembly including a shaft and a plurality of planar blades extending from the shaft; a rod including a first end and a second end; and an adjustment knob structure. Here, the impeller assembly shaft is connected to the first end of the rod, the rod extends through the adjustment knob structure, and the second end of the rod is connected to a first O-ring outside the adjustment knob structure. The impeller assembly is partially retractable, and the bioprocessing container is configured for activating and expanding T cells.

[0012] In some embodiments, the adjustment knob structure includes a series of internal helical grooves on its inner surface, the inner surface being connected to a screw, the screw including a series of helical grooves on its outer surface, and the rod extending through the screw. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body. In some embodiments, when the adjustment knob structure is released, the retracted impeller assembly remains retracted.

[0013] In some embodiments, the impeller assembly includes a magnet. In some embodiments, when the impeller assembly is in its maximum retracted state, it does not experience magnetic interference with the magnetic bead.

[0014] In some embodiments, a first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly. In some embodiments, the impeller assembly is rotatable against the rod.

[0015] According to some aspects of this disclosure, a bioprocessing container is provided, the bioprocessing container comprising: a container body including a top portion and a bottom portion; one or more necked inlet ports; an impeller assembly including a shaft and a plurality of planar blades extending from the shaft; a rod including a first end and a second end; and an adjustment knob structure. In this aspect, the shaft of the impeller assembly is connected to the first end of the rod, the second end of the rod extends through the top portion of the container body and through a first screw connected to the outer side of the top portion of the container body, and the second end of the rod is connected to an inner surface of a second screw, the second screw including a series of helical grooves on its outer surface. The second screw further includes an outer surface reversibly connected to an inner surface of the adjustment knob structure, and the second screw is connected to a spring, the spring also being connected to the adjustment knob structure. Additionally, the impeller assembly is partially retractable, and the bioprocessing container is configured for activating and expanding T cells.

[0016] In some embodiments, the flexible pouch structure is connected to the top portion of the bioprocess container via the first screw and to the inner surface of the adjustable knob structure, with the rod, the spring, and the second screw located within the flexible pouch structure. In some embodiments, the impeller assembly is retracted by connecting the second screw to the first screw and extended by disconnecting the second screw from the first screw. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body. In some embodiments, the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body. In some embodiments, the retracted impeller assembly remains retracted when the adjusting knob structure is released.

[0017] In some embodiments, the impeller assembly further includes a magnet. In some embodiments, when the impeller assembly is in its maximum retracted state, it does not experience magnetic interference with the magnetic bead.

[0018] In some embodiments, a first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly. In some embodiments, the impeller assembly is rotatable against the rod.

[0019] According to some aspects of this disclosure, a bioprocessing container is provided, the bioprocessing container comprising: a container body including a top portion and a bottom portion; one or more necked inlet ports; an impeller assembly including a shaft and a plurality of planar blades extending from the shaft; a rod including a first end and a second end; and a lever extending through one of the one or more necked inlet ports. In this aspect, the impeller assembly shaft is connected to the first end of the rod, the rod extending through a first screw, the first screw including a reversible connecting fitting for attaching a second screw to an inner surface at a center of the inner surface of the top portion of the container body, and an O-ring reversibly connecting the second screw to the second end of the rod. Additionally, the impeller assembly is partially retractable, and the bioprocessing container is configured for activating and expanding T cells.

[0020] In some embodiments, the shaft includes at least one hole into which the lever can be fitted. In some embodiments, rotating the lever in the at least one hole in a first direction disengages the first screw from the second screw, thereby extending the impeller assembly, and rotating the lever in a second direction engages the first screw with the second screw, thereby retracting the impeller assembly. In some embodiments, the impeller assembly is configured to retract from its fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body. In some embodiments, the impeller assembly is configured to retract from its fully extended position to a position between approximately one-third and approximately half the height of the container body.

[0021] In some embodiments, the impeller assembly further includes a magnet. In some embodiments, when the impeller assembly is in its maximum retracted state, it does not experience magnetic interference with the magnetic bead.

[0022] In some embodiments, a first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly. In some embodiments, the impeller assembly is rotatable against the rod.

[0023] According to some aspects of this disclosure, a method for activating and expanding T cells is provided, the method comprising the steps of: (a) providing a bioprocess container according to any of the foregoing embodiments, (b) providing the bioprocess container with an aqueous solution suitable for T cell activation and expansion, (c) providing beads to the bioprocess container, wherein the beads contain a T cell activator, (d) providing T cells to the bioprocess container, and (e) culturing the T cells activated by the beads in the bioprocess container. In some embodiments, steps (a) and (b) are performed before step (c), and step (d) is performed after step (c). In some embodiments, steps (a) and (b) are performed before step (d), and step (c) is performed after step (d). In some embodiments, the method further comprises the step (f) of mixing the aqueous solution using the impeller of the impeller assembly.

[0024] In some embodiments, the impeller includes a magnet located in the lower portion of the impeller. In some embodiments, the bead is a magnetic bead, and wherein the magnetic bead is non-magnetically attached to the impeller magnet during activation and amplification.

[0025] In some embodiments, the T cells supplied to the bioprocessing container are engineered T cells. In some embodiments, the engineered T cells contain genes for chimeric antigen receptors or T cell receptors. In some embodiments, the T cell activator is an antigen of the αβ-T cell receptor on the T cells. In some embodiments, the antigen contains anti-CD3 or anti-CD28, or a combination thereof.

[0026] Further features and advantages will be set forth in the detailed description below, and will be apparent in part from the description or to those skilled in the art by practice of the embodiments described herein, including the detailed description below, the claims and the drawings.

[0027] It should be understood that the foregoing general description and the following detailed description are merely exemplary and intended to provide an overview or framework for understanding the nature and characteristics of the claims. Drawings are included to provide further understanding; the drawings are incorporated in and form a part of this specification. The drawings illustrate one or more embodiments and, together with the description, serve to explain the principles and operation of the various embodiments. Attached Figure Description

[0028] The following is a description of the accompanying drawings, which are given purely by way of non-limiting example. The drawings are not necessarily drawn to scale, and for clarity and brevity, some features and views may be shown exaggerated to scale or in illustration.

[0029] Figure 1 This is a perspective view of an exemplary bioprocess container with an adjustment knob structure according to an embodiment.

[0030] Figure 2 This is a perspective view of an exemplary bioprocess container according to an embodiment, having an adjustment knob structure and an impeller in a non-retracted position (stationary state).

[0031] Figure 3 This is a perspective view of an exemplary bioprocess container according to an embodiment, having an adjustment knob structure and an impeller in a partially retracted position (retracted state).

[0032] Figure 4 This is a bottom view of an exemplary bioprocess container according to an embodiment, the exemplary bioprocess container having a central protrusion, planar impeller blades, and baffles on the sidewalls of the container body.

[0033] Figure 5 This is a perspective view of an exemplary bioprocess container according to an embodiment, the container having baffles on its sidewalls and an impeller in an extended position, having positioning protrusions with magnets for positioning the impeller.

[0034] Figure 6 This is an enlarged view of the exemplary intersection between a cross-sectional view of the bottom surface of the bioprocess container according to an embodiment and the lower portion of the impeller. The bottom surface includes positioning bumps and a central bump. The impeller includes planar blades, a magnet, a cutout portion on the bottom of the blades for clearing the bumps, and an O-ring connected to the cutout portion.

[0035] Figure 7 This is an exploded view of the components of a bioprocess container according to an embodiment. The bioprocess container includes a container body, an adjustment knob structure, a constricted inlet port with a cap, and an impeller assembly connected to a series of four screws for connecting the adjustment knob structure at the top of the container to the blades of the impeller.

[0036] Figure 8 This is an exploded view of the components of a bioprocess container according to an embodiment. The bioprocess container includes a container body, an adjustment knob structure, a constricted inlet port with a cap, and an impeller assembly connected to a series of three screws for connecting a cover, an O-ring, and the adjustment knob structure at the top of the container to the blades of the impeller.

[0037] Figure 9 This is an exploded view of the components of a bioprocess container according to an embodiment. The bioprocess container includes a container body, an adjustment knob structure, a constricted inlet port with a cap, an impeller assembly connected to the adjustment knob structure by a series of screws, a flexible bag, and a spring within the flexible bag.

[0038] Figure 10 This is an exploded view of the components of a bioprocess container according to an embodiment. The bioprocess container includes a container body, a mechanical lever structure, a constricted inlet port with a cap, and an impeller assembly connected to the mechanical lever structure by a series of two screws. Detailed Implementation

[0039] Various aspects and embodiments will now be described in full herein. However, these aspects and embodiments may be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the subject matter to those skilled in the art. All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0040] Modifications to this disclosure can be made by those skilled in the art and by those who have made or used this disclosure. Therefore, it should be understood that the embodiments shown in the figures and described above are for illustrative purposes only and are not intended to limit the scope of this disclosure, which is defined by the appended claims as interpreted in accordance with the principles of patent law, including the doctrine of equivalents.

[0041] A. Definition

[0042] Unless otherwise defined, all terms and phrases used herein include the meanings that have been acquired in the art, unless the opposite meaning is clearly indicated or explicitly stated from the context in which the term or phrase is used. Although methods and materials similar to or equivalent to any methods and materials described herein may be used to practice or test this disclosure, specific methods and materials are described hereafter.

[0043] As used herein, the terms “the” and “a / an” mean “at least one” and should not be limited to “only one” unless explicitly indicated otherwise. Thus, for example, unless the context explicitly indicates otherwise, references to “component” include embodiments having two or more such components.

[0044] Unless otherwise stated, a single numerical value is presented as an approximation as if the value began with the words “about” or “approximately”. Similarly, unless otherwise explicitly indicated, numerical values ​​within the various ranges specified in this application are presented as approximations as if both the minimum and maximum values ​​within the stated range began with the words “about” or “approximately”. In this way, variations above and below the stated range can achieve substantially the same results as values ​​within the range. As used herein, when referring to numerical values, the terms “about” and “approximately” should have their simple and common meaning to those skilled in the art most closely related to the disclosed subject matter or to the scope or element discussed. The amount extended from strict numerical boundaries depends on many factors. Some factors that can be considered include, for example, the criticality of the element and / or the impact of a change in a given amount on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of significant figures for different numerical values ​​does not imply a limitation on how the use of the words “about” or “approximately” will be used to extend a particular value or range. Therefore, in general, “about” or “approximately” extends the numerical value. Furthermore, the disclosure of ranges is intended as a continuous range, including every value between the minimum and maximum values, plus the extension of the range provided by the use of the terms "about" or "approximately". Therefore, references to numerical ranges herein are intended only as a shorthand way of referring to each individual value falling within said range, and each individual value is incorporated herein as if individually referenced herein.

[0045] As used herein, when used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition is described as containing components A, B, and / or C, the composition may contain only A; only B; only C; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0046] As used in this article, “have,” “having,” “include,” “including,” “comprise,” and “comprising” are all used in their open-ended sense and usually mean “including but not limited to.”

[0047] "Optional" or "optionally" means that the element, component, or condition described below may or may not appear, and the description includes examples of the element, component, or condition appearing and examples of its non-appearance.

[0048] In this document, relational terms such as first and second, top and bottom are used only to distinguish one entity or action from another, and do not necessarily need to indicate any actual such relationship or order between such entities or actions.

[0049] Unless otherwise stated, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are for ease of understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0050] B. Introduction

[0051] Rotary flasks are a low-cost option for cell culture; however, current rotary flasks lack compatibility with bead-based T-cell activation technologies. Antibody-conjugated magnetic beads are widely used to activate T cells in the production of engineered T-cell immunotherapies, but rotary flasks have magnetic impellers that interfere with bead-based T-cell activation technologies. Furthermore, the magnetic attraction and repulsion between the magnetic beads and the magnetic impeller hinders the use of antibody-conjugated magnetic beads for T-cell activation due to the magnetic attraction and repulsion forces between them.

[0052] This disclosure provides a single container for T-cell activation in patient-derived T-cell immunotherapy using all bead types (including magnetic beads), engineered T cells, and expanded modified T cells. The single container includes at least a partially retractable impeller that does not interfere with bead-based T-cell activation. The retractable impeller allows bead-based T-cell activation, including T-cell activation based on antibody-conjugated / antigen-conjugated magnetic beads, to occur within a rotary flask. The retractable impeller allows T-cell activation to be performed within the same container as the engineered T-cell and expanded modified T-cell processes. This closed-system container minimizes the risk of contamination and reduces the number of processing steps required to create engineered T-cell therapies using rotary flasks.

[0053] Further features and advantages will be set forth in the following detailed description and will be apparent to those skilled in the art, or will be recognized by practice of the embodiments described below, as well as the claims and drawings.

[0054] C. Bioprocess containers

[0055] refer to Figure 1-3According to aspects of this disclosure, an exemplary bioprocessing container 6 for engineering, activating, and expanding T cells is shown. The bioprocessing container 6 includes a container body 10, at least one constricted inlet port (e.g., 16a, 16b), at least one cap (e.g., 14a, 14b), an impeller assembly 30, and an adjustment knob structure 18 that allows the impeller assembly 30 to translate vertically along the central axis of the bioprocessing container 6. The container body 10 further includes a top portion 7 and a bottom portion 8, the top portion surrounding an end of the bioprocessing container 6 having the adjustment knob structure 18, and the bottom portion surrounding an end of the bioprocessing container 6 opposite to the top portion. In some embodiments, the bioprocessing container 6 has a baffle 12 projecting toward the interior of the bioprocessing container, as shown in… Figure 1 and 4 As can be seen in the text. The size range of bioprocessing containers is typically from about 125 mL to about 50 liters, but smaller or larger sizes are also considered. In one embodiment, the size of the bioprocessing container is about half a liter, about one liter, about two liters, about three liters, about five liters, about 10 liters, about 20 liters, about 25 liters, about 35 liters, or about 50 liters.

[0056] The impeller assembly 30 of this disclosure includes an impeller blade arrangement 20 and an impeller shaft 26 extending along a vertical axis from an adjustment knob structure 18 toward the bottom of the bioprocess container 6. The shaft 26 may be flexible, rigid, solid, or have other varying degrees of flexibility, such as a series of helical grooves in its tip. Extending from the shaft 26 is the blade arrangement 20, which extends toward the bottom of the bioprocess container 6. The blade arrangement 20 comprises a plurality of planar blades. In one embodiment, the impeller assembly 30 includes a shaft 26 and a plurality of planar blades extending from the shaft 26. Figure 2-3 In the illustrated embodiment, the blade arrangement 20 has four planar blades, each planar blade positioned 90 degrees relative to each other. Of the four planar blades, there are two main blades 50 and two auxiliary blades 54. The main blades 50 are positioned 180 degrees relative to each other, and similarly, the two auxiliary blades 54 are positioned 180 degrees relative to each other. The auxiliary blades are smaller than the main blades. The blade arrangement 20 of main and auxiliary blades around axis 26 produces an alternating effect of auxiliary-main blade orientation. However, as those skilled in the art will understand, other blade arrangements may be employed in this disclosure, including arrangements with fewer or more than four blades, and blade arrangements with different shapes and sizes. For example, a blade arrangement may contain two blades of the same or unequal sizes. In another example, a blade arrangement may contain three blades, each with a different shape.

[0057] refer to Figure 4In some embodiments, an impeller O-ring 40 is located at the bottom of shaft 26 and below the main blade 50 and secondary blade 54 of the impeller assembly. The impeller O-ring 40 is coupled to the bottom cutout portion of the planar blades. In some embodiments, the bottom edge of the impeller O-ring is flush with the bottom edges of the planar blades 50, 54. In other embodiments, the bottom edge of the impeller O-ring is located below the bottom edges of the planar blades 50, 54. A plurality of locating bumps 38 are coupled to the bottom inner surface of the impeller O-ring 40, spaced apart from or along the inner edge 40a of the impeller O-ring 40. In some embodiments, a set of locating bumps may have two, three, four, five, six, or more locating bumps 38 spaced apart from each other along the inner edge 40a of the impeller O-ring 40. In some embodiments, the plurality of locating bumps 38 may be equally spaced from each other along the inner edge 40a of the impeller O-ring 40. In one embodiment, the plurality of locating bumps is three locating bumps 38, each locating bump being equally spaced from each other. The multiple positioning bumps may have each bump spaced equidistant from each other along the inner edge 40a of the impeller O-ring 40, or may have each bump spaced unequally from each other. Figure 4 The baffle 12 of the bioprocess container is further depicted protruding inward from the container body 10.

[0058] In some embodiments, the plurality of positioning bumps 38 may comprise cylindrical (e.g., Figure 5 (Ref. 96 in the attached figures), blocky, square, conical (e.g., Figure 6 (Ref. 94 in the accompanying drawings) A single protrusion in the shape of a rectangle or pyramid. Multiple locating protrusions 38 may also have other shapes or combinations of shapes. In some respects, the shape of the locating protrusion 38 may be configured to make constant, periodic or frequent contact with the inner edge 40a of the impeller O-ring 40 to prevent vertical or lateral movement of the impeller assembly.

[0059] In some embodiments, the bottom inner surface of the bioprocessing container has a central bump 42. The central bump 42 is a raised feature, such as... Figure 5-6 As shown, it can contain various shapes. For example, the central bump 42 can be cylindrical (see...). Figure 5 ), blocky, square, conical (see Figure 6 The center bump 42 may be rectangular, pyramidal, or any other shape. In some embodiments, the center bump 42 may have rounded or beveled edges. The center bump 42 serves at least two purposes: (1) to prevent cells and / or beads from converging or aggregating beneath the impeller assembly; and (2) to minimize vertical or lateral movement of the impeller assembly during transport and rotation to avoid impeller breakage. While the raised center bump 42 helps minimize vertical or lateral movement of the impeller assembly, the positioning bump 38 also helps minimize both vertical and lateral movement of the impeller assembly.

[0060] The impeller assembly of this disclosure can sometimes be positioned in a stationary state (i.e., non-retracted positioning), and the bioprocessing container can be used for T-cell engineering and expansion during the stationary state. In the stationary state, in some embodiments, the distance between the top surface of the central bump 42 and the bottom of the impeller assembly can be from about 0.001 inches to about 0.1 inches. In other embodiments, the distance between the top of the central bump 42 and the bottom of the impeller assembly can be from about 0.005 inches to about 0.05 inches. In still other embodiments, the distance between the top of the central bump 42 and the bottom of the impeller assembly can be from about 0.01 inches to about 0.03 inches. For example, the distance between the top of the central bump 42 and the bottom of the impeller assembly can be about 0.0150 inches, about 0.0185 inches, about 0.0200 inches, about 0.0250 inches, or all distance values ​​between these referenced values ​​and ranges disclosed herein. In some embodiments, the tolerances for the plurality of positioning bumps 38, center bump 42, impeller assembly, and other dimensional features of the bioprocess container are from ±0.001 inches to ±0.100 inches. In one specific embodiment, the tolerances for the plurality of positioning bumps 38, center bump 42, impeller assembly, and other dimensional features of the bioprocess container are approximately ±0.005 inches.

[0061] The impeller assembly of this disclosure can sometimes be positioned in a retracted state, and the bioprocess container can be used for bead-based T-cell activation during the retracted state. In the retracted state, the impeller assembly is retracted from the bottom inner surface of the bioprocess container such that the impeller assembly does not interfere with the bead-based T-cell activation process. The distance between the top surface of the central bump 42 and the bottom of the impeller assembly depends on the size of the bioprocess container and the volume of culture medium during the T-cell activation step. In the retracted state, in some embodiments, the impeller assembly is retracted from the bottom inner surface of the bioprocess container, where the impeller assembly is in its fully extended position up to a distance between approximately 1 / 6 (one-sixth) and approximately 2 / 3 (two-thirds) of the height of the container body 10. The height of the container body includes the top and bottom portions of the container body but excludes any adjustment knob structures for vertical translation of the impeller assembly and any necked-in ports on the top portion of the container body. In some embodiments, the impeller assembly is retracted from the bottom inner surface of the bioprocess container up to a distance between approximately 1 / 3 (one-third) and approximately 1 / 2 (half) of the height of the container body 10.

[0062] In the stationary or retracted state, the distance between the base outer diameter of the central protrusion 42 (the diameter of the central protrusion connected to the bottom inner surface) and the inner diameter of the impeller O-ring 40 can be from about 0.01 inches to about 1.0 inch. In other embodiments, the distance between the base outer diameter of the central protrusion 42 and the inner diameter of the impeller O-ring 40 can be from about 0.1 inches to about 0.5 inches. In still other embodiments, the distance between the base outer diameter of the central protrusion 42 and the inner diameter of the impeller O-ring 40 can be from about 0.2 inches to about 0.3 inches. For example, the distance between the base outer diameter of the central protrusion 42 and the inner diameter of the impeller O-ring 40 can be about 0.15 inches, about 0.20 inches, about 0.025 inches, about 0.030 inches, about 0.035 inches, or any value or range between 0.01 inches and 1.0 inch.

[0063] The bioprocessing container for culturing cells disclosed herein provides an readily available, affordable, disposable, pre-sterilized, fully integrated cell culture vessel that offers gentle agitation to minimize hydrodynamic shear and maintain cell suspension within the bioprocessing container. A central bump and / or positioning bump on the bottom inner surface of the bioprocessing container prevents bead and / or cell aggregation below the impeller assembly and minimizes vertical movement of the impeller assembly during transport and rotation to prevent damage to the beads, cells, and / or the impeller assembly.

[0064] Regarding the impeller size, the impeller assembly 30 can be sized such that the blades 50, 54 extend almost to the full diameter of the bioprocess container 6. In one embodiment, the impeller blades 50, 54 extend approximately 50-95% of the radius of the bioprocess container, as measured from the midpoint of the cross-sectional area between the vertical sidewalls inside the container body. In another embodiment, at least one blade 50, 54 extends 75-95% of the radius of the bioprocess container, as measured from the midpoint of the cross-sectional area between the vertical sidewalls inside the container body. However, it should be understood that the impeller blades can extend any distance, including less than 50% of the radius of the bioprocess container, or even greater than 95% but less than 100% of the radius of the bioprocess container, as measured from the midpoint of the cross-sectional area between the vertical sidewalls inside the container body.

[0065] The bottom portion of the impeller assembly (the end closest to the bottom of the bioprocess vessel) has a cutout formed within the intersection of the bottom edges of the corresponding impeller blades. The bottom cutout substantially follows the contour of the central bump. For example, if the central bump is cylindrical, the bottom cutout substantially follows the cylindrical shape of the central bump, such as... Figure 5 As shown. As another example, if the center bump is conical in shape, the bottom cutout portion essentially follows the conical shape of the center bump, such as... Figure 6 As shown.

[0066] When the bioprocessing vessel is positioned vertically, the impeller O-ring attached to the bottom cutout portion of the impeller assembly is not intended to contact the central bump. In some embodiments, there may be no contact point between the impeller assembly and the bioprocessing vessel below the impeller shaft housing. This is advantageous because it helps reduce the likelihood of cell damage should cells or beads be trapped between the bottom edge of the planar impeller blades and the central bump, or due to shear stress. The central bump on the bottom inner surface 70 of the bioprocessing vessel also eliminates any potential dead points (the point of least turbulence generated by the rotating impeller) directly below the central axis of the impeller assembly. The bottom cutout portion within the intersection of the impeller blades 50, 54 allows the blade edge 66 to be in close proximity to the bottom inner surface 70, such as... Figure 6 As shown in the enlarged view, in one embodiment, the distance between the impeller blades 50, 54 and the bottom inner surface 70 of the bioprocess container is between approximately 0.05 inches and approximately 0.5 inches. Since the bioprocess container is intended to be transported as a single unit, the impeller O-ring, which engages with the central bump, also serves to accommodate the impeller assembly during transport, preventing the impeller assembly from disengaging from the bottom of the bioprocess container or from damaging the sidewalls of the container body due to contact caused by the pushing of the bioprocess container.

[0067] In some embodiments, the impeller has a magnet located in the bottom portion of the impeller. For example... Figure 4 and Figure 6 As shown, a magnet housing 24 for receiving a magnetic stirring rod (magnet) 22 is molded into the lower portion of each of the two main blades 50. Holes in the regions of the secondary blades 54 and shaft 26 complete the magnet housing 24. A cylindrical plug or magnetic stirring rod 22 is mounted in the magnet housing 24 along the lower edges of the two main blades 50 and orthogonal to the secondary blades 54. Alternatively, the magnet 22 itself is molded into the impeller assembly. To achieve this, the magnet 22 is inserted into a mold, and the impeller assembly is molded around the magnet 22 itself. Molding the magnet 22 integrally within the impeller assembly provides the advantage that the magnet 22 will not detach from the impeller assembly and damage the bioprocess vessel during assembly and transport.

[0068] refer to Figure 7In some embodiments, the rod 100 has a first end and a second end, the first end having an O-ring (not shown), and the second end having a series of helical grooves 101. The rod 100 is positioned within the inner channel of the central shaft 26 such that the blade arrangement 20 is attached to and can rotate freely against the end of the rod 100 containing the O-ring, while the end of the rod containing the helical grooves extends from the top of the central shaft 26. For the impeller assembly 30, the rod 100 is secured by a first screw structure 110 having a series of helical grooves on its top portion, a port 16c of the top part 74 of the top portion 7 of the container body 10, a second screw structure 111 having a series of helical grooves in its channel, a third screw structure 112, an adjustment knob structure 18 having a series of helical grooves in its channel, and finally a fourth screw structure 113. The first screw structure 110 engages with the inner helical grooves of the second screw structure 111 and can be fastened to the top part 74 of the top portion 7 of the container body 10. The third screw structure 112 attaches the adjustment knob structure 18 to the top part 74 of the top portion 7 of the container body 10 to establish a closed system. The fourth screw structure 113 mates with a helical groove at the tip of the rod 100 and holds the entire impeller assembly in place, allowing the entire impeller assembly to be adjusted to a predetermined height (i.e., stationary or retracted) from the bottom of the bioprocess container 6 by tightening or loosening the fourth screw structure 113. Tightening retracts the impeller assembly, while loosening lowers it. Additional O-rings can be placed between different layers or structures to form a tight seal for an even tighter closed system. In some embodiments, the third screw structure 112 is absent, and the adjustment knob structure 18 contacts the second screw structure 111 directly to form a tight seal. In some embodiments, the adjustment knob structure 18 and the fourth screw structure 113 are enclosed within a soft pouch structure, so that the entire container remains a closed system during the operation of the T-cell activation and amplification process, where the impeller assembly needs to retract first and then not retract.

[0069] refer to Figure 8In some embodiments, the rod 100 has a first end with an O-ring (not shown) positioned within an inner channel of the central shaft 26, such that the blade arrangement 20 is attached to the first end of the rod 100 and can rotate freely against the rod 100. A second end of the rod 100 extends from the central shaft 26, passing through a first screw structure 110 having a series of helical grooves on its top portion, a port 16c of the top part 74 of the top portion 7 of the container body 10, a second screw structure 111 having a series of helical grooves within its channel, a third screw structure 112 having a series of helical grooves on its exterior, an adjustment knob structure 18 having a series of helical grooves within its channel, an O-ring 121 attached to the second end of the rod 100, and finally through a cover 18a to form a closed system. The O-ring 121 in the second end of the rod 100 allows the entire impeller assembly to be attached to the adjustment knob structure 18. The first screw structure 110 engages with the inner helical grooves of the second screw structure 111 and can be fastened to the top part 74 of the top portion 7 of the container body 10. The outer helical groove of the third screw structure 112 matches the inner helical groove of the second screw structure 111 and the inner helical groove of the adjusting knob structure 18. In this way, by tightening or loosening the adjusting knob structure 18 against the third screw structure 112, the entire impeller assembly can be adjusted from a position near the bottom of the bioprocess container 6 to a predetermined height (i.e., a stationary or retracted state). Tightening causes the impeller assembly to retract, while loosening causes it to lower. Additional O-rings can be placed between different layers or structures to create a better seal, resulting in an even tighter closed system. For the O-rings described in any of the embodiments herein, the O-rings can be made of PTFE (polytetrafluoroethylene), nylon, or other similar low-friction materials.

[0070] refer to Figure 9In some embodiments, the rod 100 has a first end with an O-ring (not shown) positioned within an inner channel of the central shaft 26, such that the blade arrangement 20 is attached to the first end of the rod 100 and can rotate freely against the rod 100. A second end of the rod 100 extends from the central shaft 26 via a first screw structure 110 having a series of helical grooves on its top portion, a port 16c of the top part 74 of the top portion 7 of the container body 10, the bottom of the flexible bag-like structure 120, a second screw structure 111 having a series of helical grooves within its channel, a third screw structure 112 having a series of helical grooves on its exterior, an O-ring attached to the second end of the rod 100, a spring 115, the top of the flexible bag-like structure 120, and an adjustment knob structure 18 having a series of helical grooves within its channel. The first screw structure 110 engages with the inner helical grooves of the second screw structure 111 and can be fastened to the top part 74 of the top portion 7 of the container body 10. The inner helical groove of the adjusting knob structure 18 matches the outer helical groove of the third screw structure 112, attaching the entire impeller assembly to the adjusting knob structure 18. When the third screw structure 112 connects the adjusting knob structure to the top part 74 of the top portion 7 of the container body 10, the spring 115 is compressed, bringing the entire impeller assembly to a stationary state (i.e., closest to the bottom of the bioprocess container 6). When the third screw structure 112 disengages from the second screw structure 111 (i.e., from the top part 74 of the top portion 7 of the bioprocess container 6), the spring relaxes, and the entire impeller assembly is in a retracted state (i.e., the impeller assembly is away from the bottom of the bioprocess container 6). The flexible bag-like structure 120 ensures that the entire bioprocess container is a closed system, regardless of the state of the impeller assembly. Additionally, O-rings can be placed between different layers or structures to create a better seal, resulting in a tighter closed system.

[0071] refer to Figure 10In some embodiments, the rod 100 has a first end with an O-ring and is positioned within an inner channel of the central shaft 26, such that the blade arrangement 10 is attached to the first end of the rod 100 and can rotate freely against the rod 100. A second end of the rod 100 extends from the central shaft 26 via a first screw structure 110 having a series of helical grooves on its top portion and an O-ring 118 attached to the second end of the rod 100. The O-ring is smaller than the diameter of the top of the first screw structure 110 and serves as a mechanical component for holding the impeller assembly against the first screw structure 110, while the O-ring does not interfere with the insertion of the first screw structure 110 into the second screw structure 111. The first screw structure 110 is then inserted into the second screw structure 111 by rotation to secure them together, wherein the second screw structure 111 is fixed in its central position below the top portion 7 of the container body 10, such that the entire impeller assembly is attached to the top portion of the container. When the impeller assembly is stationary (i.e., near the bottom of the bioprocess container 6), lever 120 is inserted into the constricted inlet port 16a through its cover 14a and is freely suspended. When the second end of lever 120 is inserted into a hole in the side of the top of the central shaft 26 and rotated, the impeller assembly can retract from the bottom of the bioprocess container 6. An O-ring can be placed between the first and second screw structures to form a better seal, resulting in a tighter closed system.

[0072] The maximum height achievable above the bottom of the inner surface of the bioprocess vessel depends on the position where the impeller blades on the impeller shaft begin to extend from the shaft, and the design of the bioprocess vessel. If the blades themselves are too large to pass through the threads on the knob housing, then the maximum height above the bottom of the inner surface will be equal to or slightly less than the position where the blades extend from the shaft. When according to Figure 7 When assembling the bioprocess container, the maximum retraction height also depends on the length of the helical groove 101 at the second end of the rod 100. When according to... Figure 8 When assembling the bioprocess container, the maximum retraction height also depends on the length of the third screw structure 112. When according to... Figure 9 When assembling the bioprocess container, the maximum retraction height also depends on the relaxation length of the spring structure 115. In contrast, when based on... Figure 10 When assembling the bioprocessing vessel, the maximum retraction height also depends on the distance that lever 120 can lift the impeller assembly. Additionally, when the impeller assembly is retracted from the culture medium, a static state is achieved (i.e., the blades are not rotating in the fluid), and the magnetic force between the magnetic beads and any magnets on the impeller assembly is reduced or eliminated. This allows magnetic beads carrying T-cell activators to be used with the bioprocessing vessel. In some embodiments, no magnetic interference occurs between the magnetic beads and the impeller magnets when the impeller assembly is retracted to its maximum extent. As used herein, “magnetic interference” means that the magnetic beads are magnetically attached to the impeller magnets.

[0073] like Figure 1-3 As shown, one or more necked-in ports 16 extend outward from the top portion 7 of the bioprocess container 6. In some embodiments, one or more caps 14 may be removably attached to the necked-in ports 16. In one embodiment, at least one internally threaded cap is removably attached to an externally threaded necked-in port. In some embodiments, as Figure 7-10 As shown, at least one cap 14 has a vent 75 that allows necessary gas communication with the external environment. In some embodiments, the cap has internal threads and is removably attached to a necked inlet port with external threads, and the cap further includes a vent. In another embodiment, fittings such as tubes may be used and connected to one or more necked inlets 16 to allow aseptic dispensing. In another embodiment, as... Figure 7 As shown in the example, the cover 14 includes a hydrophobic membrane insert 80 made of a material that allows gas to be transported into the interior of the bioprocess vessel, but prevents liquid from escaping from the bioprocess vessel 6 and other contaminants from entering the bioprocess vessel 6. Examples of such membrane materials include polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF).

[0074] In some embodiments, such as Figure 1-3 As illustrated in examples 7-10, one or more constricted inlet ports 16 extend at an angle to the horizontal direction to allow instruments such as pipettes to pass through the impeller assembly and reach adjacent areas of the stirred vessel at a pre-selected depth. However, the size of one or more inlet ports 16 and the angle at which the inlet ports 16 extend from the vessel body 10 can be selected to optimize instrument accessibility to areas within various bioprocessing vessels 6. Additionally, in some embodiments, one or more inlet ports are two inlet ports 16, such as... Figure 1-3 As disclosed in the examples in 7-10, but it should be understood that any number of port 16 is possible.

[0075] In some embodiments, as shown in the figure, a plurality of baffles 12 extend along the inner wall of the container body 10 in a vertical direction parallel to the central axis of the bioprocess container 6. In some embodiments, as shown in the figure, the cross-sectional shape of the baffles 12 is approximately semi-cylindrical or isosceles triangle. Each baffle 12 originates from the bottom of the bioprocess container and extends vertically upward, terminating in an elliptical shape (see Figure 12). Figure 5(Ref. 56 in the accompanying drawings). Although the baffle 12 shown herein is depicted as terminating in an elliptical shape, embodiments of this disclosure are not limited thereto and may take any shape. It is believed that extending the plurality of baffles 12 completely through the liquid region (i.e., from the bottom inner surface 70 of the container body 10 to a point above the liquid surface) enhances turbulence throughout the liquid region. The plurality of baffles 12 extend into the cavity of the bioprocess container and, in combination with the impeller assembly 30, generate and enable turbulence within the bioprocess container. The plurality of baffles 12 are preferably integrally formed with the wall of the container. In some embodiments, the plurality of baffles 12 are three baffles 12, each baffle spaced equidistantly from each other around a perimeter defined by the cylindrical sidewall of the container body 10. In other respects, the plurality of baffles are symmetrically arranged along the inner cylindrical sidewall of the container body 10, but the number and density of the baffles 12 may vary based on the size of the bioprocess container. In embodiments, each baffle is integrally formed with the sidewall of the container body, which originates from the bottom inner surface and extends vertically to a predetermined distance above the sidewall.

[0076] In some embodiments, the bioprocess container of this disclosure is made of an injection-molded polymer (e.g., polystyrene, polycarbonate, or any other suitable polymer identified by those skilled in the art). In one embodiment, the polymer is optically transparent and non-cytotoxic. Because the material is made of a lightweight polymer and the bioprocess container is pre-sterilized during preparation, the bioprocess container itself is disposable, and the end user does not need to sterilize the components of the system before use. In some embodiments, the body of the container body 10 of this disclosure (i.e., the bottom portion 8) is made of glass, and the top portion 7 of the container body 10 is made of plastic.

[0077] In describing the fabrication and assembly process, the impeller assembly 30, the top portion 7, and the bottom portion 8 of the container body 10 can be molded separately and processed as discussed. Subsequently, in embodiments where the magnet 22 is not overmolded, the magnet 22 is placed in the magnet receiving portion 24. In embodiments where the magnet 22 itself is overmolded and thus integral with the blade arrangement 20, the magnet is added during the molding stage. Then, according to... Figure 7-9In the illustrated embodiment, the blade assembly 20 is attached to the rod 100, which is connected to the top portion 7 of the container body 10 via an adjustment knob structure 18. An O-ring 40 slides over the end of the impeller shaft 26 and contacts a receiving groove. In some embodiments, the top portion 7 and the bottom portion 8 of the container body 10 are then permanently fixed together by, for example, ultrasonic welding along a weld line, thereby creating a fully and permanently integral unit. In other embodiments, the parts are laser welded or attached by adhesive. In embodiments with one or more necked inlets 16 and a cap 14, the cap 14 is placed in place, and the unit is effectively sealed for transport. The integral unit can then be sterilized. In some embodiments, the top portion 7 is screwed to the bottom portion 8 of the container to form a tight seal. Since most cell culture procedures are performed under aseptic conditions by implementing so-called aseptic techniques, the pre-sterilization of the bioprocess container 6 provides a culture chamber to be maintained in a sterile, closed environment. Advantageously, cell culture processes are performed in a system where the culture chamber is functionally closed to the external environment, where sterile integrity is maintained from the time the container is prepared until it is discarded. One pre-sterilization method includes gamma irradiation. Other sterilization methods known to those skilled in the art may also be used, including ethylene oxide or electron beam irradiation.

[0078] The bioprocessing container of this disclosure allows for the engineering, activation (and expansion) of engineered T cells. Activation is a step that currently available rotary flasks cannot successfully perform due to interference between the impeller blades and the beads carrying the T cell activator. The bioprocessing container of this disclosure solves this problem by providing a rotary flask that can successfully perform T cell activation using the beads. This disclosure provides a retractable impeller assembly that does not interfere with the beads during activation when the impeller assembly is retracted from the bottom surface of the container. Methods for activating and expanding engineered T cells using the bioprocessing container of this disclosure will now be described.

[0079] Methods for activating and expanding T cells generally include the following steps: (1) providing a bioprocessing container of the present disclosure, (2) providing the bioprocessing container with an aqueous solution suitable for T cell activation and expansion, (3) providing the bioprocessing container with a T cell activator, (4) providing the bioprocessing container with T cells, (5) culturing the bead-activated T cells in the bioprocessing container while the impeller assembly retracts from the solution, and (6) culturing the activated T cells under rotational culture conditions to expand the cells after the impeller assembly is reset to a stationary state and rotated. Any embodiment of the bioprocessing container described above will be used to activate and expand T cells, including engineered T cells.

[0080] Aqueous solutions used for T cell activation and expansion are well known to those skilled in the art of T cell therapy. Non-limiting examples include various cell culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco). ® HyClone ® RPMI 1640 medium (Cytiva) ® Iscov's Modified Dulbecc's Medium (IMDM), OpTmizer ® CTS ® Serum-free culture medium for T cell expansion (Gibco), CTS ® AIM-V ® Culture medium (Gibco) ® X-VIVO ® 15 (Lonza) ® ) and StemSpan ® Serum-free amplification medium or StemSpan ® SFEM II medium (StemSpan, Canada) ® StemSpan Technology Company ® Technologies Canada, Inc.) and other similar culture media, including serum-containing and serum-free variants, as well as other supplemental variants (e.g., with the addition of L-glutamine, glucose, etc.).

[0081] T-cell activators are agents that stimulate T cells to initiate targeted intracellular signals, increasing the desired outcome (e.g., overexpression of certain receptors on the exterior of engineered T cells). Common examples include, but are not limited to, antigens of αβ-T cell receptors (e.g., anti-CD3 monoclonal antibodies, anti-CD28 monoclonal antibodies, anti-CD19 monoclonal antibodies, and other similar antigens), phytohemagglutinin (PHA) mitogens, certain cytokines (such as IL-2 (interleukin-2)), and combinations thereof. Any T-cell activator known to those skilled in the art can be provided to the bioprocessing container disclosed herein. To generate T-cell signaling that extends sufficiently to produce a productive response, in some embodiments, the T-cell activator may be surface-bound to mimic cell interactions. In one embodiment, the surface-bound T-cell activator may be bead-based, meaning that the T-cell activator is coated or attached to the outer surface of a bead that can be provided to the bioprocessing container. In one specific embodiment, the bead is a magnetic bead. In another specific embodiment, the bead is a polystyrene bead. In yet another specific embodiment, the bead is a glass bead. As understood by those skilled in the art of T-cell therapy, any known method of attaching an activator to the surface of a bead can be used.

[0082] The T cells supplied to the container can be any type of T cell. In some embodiments, the T cells are engineered T cells. Engineered T cells include T cells transduced with a viral vector carrying a desired gene, such as CAR or TCR. However, any genetically modified T cell is an engineered T cell. In some embodiments, the T cells are unengineered T cells. Non-limiting examples of such T cells include patient-derived CD3+ T cells or other patient-derived T cells.

[0083] To operate the bioprocess vessel of this disclosure for T-cell activation and expansion, an aqueous solution, T cells, and a T-cell activator are all added to the bioprocess vessel. The first step of the method is to provide the bioprocess vessel of this disclosure. The next step of the method is to provide the aqueous solution, T cells, and T-cell activator to the bioreactor vessel in any order. In one embodiment, the aqueous solution is provided before the T cells, and the T cells are provided before the T-cell activator. In another embodiment, the aqueous solution is provided before the T-cell activator, and the T-cell activator is provided before the T cells. In yet another embodiment, the T cells are provided before the T-cell activator, and the T cells are provided before the aqueous solution. In yet another embodiment, the T cells are provided before the aqueous solution, and the aqueous solution is provided before the T cells. Any other ordering of the aqueous solution, T cells, and T-cell activator may be used.

[0084] However, in embodiments where beads are used as T-cell activators, the impeller assembly of the bioprocessing vessel should be retracted before the beads are supplied to prevent damage to the beads or any coating on them. In embodiments using magnetic beads, retracting the impeller before adding the beads also has the advantage of reducing or eliminating magnetic interference with the beads, so they do not attract or adhere to any magnets in the impeller assembly.

[0085] T cells, T cell activators, and an aqueous solution (such as culture medium) are delivered through one or more inlet ports 16 of the container. The aqueous liquid is added until the desired level is reached in the bioprocess container (i.e., bioprocess container 6 in the figure). Preferably, the level is lower than the top edges of the blades 50, 54 and the top of the baffle 12, but higher than the lower portion of the main blade 50 (where the blade extends furthest outward from the center).

[0086] In embodiments where the impeller assembly includes a magnet, a magnetic stirring device (not shown) can be used with the bioprocess container 6 once the aqueous liquid is in the container, and the stirring device rotates the magnetic stirring rod 22 within the container. As a result, the impeller assembly 30, including the shaft 26 and blades 50, 54, also rotates within the container. The rotation of the blade arrangement 20 agitates the fluid within the container. Alternatively, the blade assembly 20 (with or without a magnet) can be rotated by a motorized mechanism engaged with the top of the shaft 26. The shape of the blades 50, 54 and their interaction with the baffle 12 cause the liquid to circulate from a position near the top of the liquid level to a position near the bottom of the liquid level. The central bump 42 prevents material buildup at the center of the bottom inner surface 70 of the bioprocess container 6. Because the upper portion 52 of the main blade 50 extends above the liquid level, the surface area of ​​the liquid in the container is effectively increased and continuously agitated, resulting in aeration of the liquid. This fluid agitation similarly occurs in any other blade configuration, including blade configurations with only one type of blade or blades with different shapes.

[0087] The impeller assembly of the bioprocessing vessel is used to agitate T cells after activation. Preferably, mixing is efficient, allowing the liquid to circulate from the bottom of the device to the surface and back again. Typically, the cells are maintained at approximately 27-37°C and mixed at 5 rpm to 300 rpm. However, as those skilled in the art will understand, these conditions can vary depending on the specific T cells, T cell activator, or application. In embodiments where magnetic beads are used as T cell activators and the impeller assembly has magnets, the maximum speed used for the impeller assembly can be lower than the maximum speed when using a non-magnetic impeller or non-magnetic beads. This is to prevent the magnetic beads from rising to a height where the magnetic attraction would cause the beads to magnetically attract and attach to the retracted impeller. In one specific embodiment, the aqueous solution is mixed at less than approximately 275 rpm, approximately 250 rpm, approximately 225 rpm, approximately 200 rpm, approximately 175 rpm, 150 rpm, 125 rpm, 100 rpm, 75 rpm, 50 rpm, or 25 rpm, and the magnetic beads are non-magnetically attached to the retracted impeller.

[0088] The activation and amplification process may be carried out over a relatively long period of time. In one embodiment, the activation and amplification process may be carried out between 2 hours and 3 months. In another embodiment, the activation and amplification process may be carried out between 0.5 days and 10 days. In some embodiments, the used aqueous solution can be replaced with fresh aqueous solution through a constricted inlet port on the bioprocess vessel. This is advantageous for activation and amplification processes that typically require several days or weeks, as it allows optimal conditions to be maintained throughout the process.

[0089] T cells or cell material can be collected via a constricted inlet port using a pipette, pour, or pump. When collecting T cells, the impeller assembly can retract or extend, or be positioned somewhere in between.

[0090] While this disclosure includes a limited number of embodiments, those skilled in the art who benefit from this disclosure will understand that other embodiments can be devised without departing from the scope of this disclosure.

Claims

1. A bioprocess container, comprising: A container body, the container body comprising a top portion and a bottom portion; One or more necked-in ports; An impeller assembly comprising a shaft and a plurality of planar blades extending from the shaft; A rod, the rod comprising a first end and a second end; and Adjust the knob structure; The shaft of the impeller assembly is connected to the first end of the rod, the rod extends through the adjustment knob structure, and the second end of the rod is connected to a screw outside the adjustment knob structure; The impeller assembly described therein is partially retractable; and The bioprocess container is configured to activate and expand T cells.

2. The bioprocessing container of claim 1, wherein the second end of the rod includes a series of helical grooves connected to an internal helical groove on the screw.

3. The bioprocess container of claim 1, wherein the impeller assembly is configured to retract from its fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body.

4. The bioprocess container of claim 3, wherein the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body.

5. The bioprocess container of claim 1, wherein the impeller assembly comprises a magnet.

6. The bioprocess container of claim 2, wherein the retracted impeller assembly remains retracted when the screw is loosened.

7. The bioprocess container according to claim 5, wherein when the impeller assembly is in its maximum retracted state, it does not cause magnetic interference with the magnetic beads.

8. The bioprocess container of claim 1, wherein the first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly.

9. The bioprocess container of claim 8, wherein the impeller assembly is rotatable against the rod.

10. A bioprocess container, comprising: A container body, the container body comprising a top portion and a bottom portion; One or more necked-in ports; An impeller assembly comprising a shaft and a plurality of planar blades extending from the shaft; A rod, the rod comprising a first end and a second end; and Adjust the knob structure; The impeller assembly shaft is connected to the first end of the rod, the rod extends through the adjustment knob structure, and the second end of the rod is connected to a first O-ring outside the adjustment knob structure; The impeller assembly described therein is partially retractable; and The bioprocess container is configured to activate and expand T cells.

11. The bioprocess container of claim 10, wherein the adjusting knob structure includes a series of internal helical grooves on its inner surface, the inner surface being connected to a screw, the screw including a series of helical grooves on its outer surface, and wherein the rod extends through the screw.

12. The bioprocess container of claim 10, wherein tightening the adjustment knob structure on the screw extends the impeller assembly, and loosening the adjustment knob structure on the screw retracts the impeller assembly.

13. The bioprocess container of claim 10, wherein the impeller assembly is configured to retract from its fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body.

14. The bioprocess container of claim 13, wherein the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body.

15. The bioprocess container of claim 10, wherein the impeller assembly comprises a magnet.

16. The bioprocess container of claim 10, wherein the retracted impeller assembly remains retracted when the adjustment knob structure is released.

17. The bioprocess container of claim 15, wherein when the impeller assembly is in its maximum retracted state, it does not cause magnetic interference with the magnetic beads.

18. The bioprocess container of claim 10, wherein the first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly.

19. The bioprocess container of claim 18, wherein the impeller assembly is rotatable against the rod.

20. A bioprocess container, comprising: A container body, the container body comprising a top portion and a bottom portion; One or more necked-in ports; An impeller assembly comprising a shaft and a plurality of planar blades extending from the shaft; A rod, the rod comprising a first end and a second end; and Adjust the knob structure; The shaft of the impeller assembly is connected to the first end of the rod, the second end of the rod extends through the top portion of the container body and through a first screw connected to the outside of the top portion of the container body, and the second end of the rod is connected to the inner surface of a second screw, the second screw having a series of helical grooves on its outer surface; The second screw further includes an outer surface that is reversibly connected to the inner surface of the adjustment knob structure, and wherein the second screw is connected to a spring that is also connected to the adjustment knob structure; The impeller assembly described therein is partially retractable; and The bioprocess container is configured to activate and expand T cells.

21. The bioprocess container of claim 20, wherein the flexible bag-like structure is connected to the top portion of the bioprocess container by the first screw and to the inner surface of the adjustable knob structure, and wherein the rod, the spring, and the second screw are located within the flexible bag-like structure.

22. The bioprocess container of claim 20, wherein the impeller assembly is retracted by connecting the second screw to the first screw, and the impeller assembly is extended by disconnecting the second screw from the first screw.

23. The bioprocess container of claim 20, wherein the impeller assembly is configured to retract from its fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body.

24. The bioprocess container of claim 23, wherein the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body.

25. The bioprocess container of claim 20, wherein the impeller assembly further comprises a magnet.

26. The bioprocess container of claim 20, wherein the retracted impeller assembly remains retracted when the adjustment knob structure is released.

27. The bioprocess container of claim 25, wherein when the impeller assembly is in its maximum retracted state, it does not cause magnetic interference with the magnetic beads.

28. The bioprocess container of claim 20, wherein the first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly.

29. The bioprocess container of claim 28, wherein the impeller assembly is rotatable against the rod.

30. A bioprocess container, comprising: A container body, the container body comprising a top portion and a bottom portion; One or more necked-in ports; An impeller assembly comprising a shaft and a plurality of planar blades extending from the shaft; A rod, the rod comprising a first end and a second end; and A lever that extends through one of the one or more constricted entry ports; The impeller assembly shaft is connected to the first end of the rod, the rod extending through a first screw, the first screw including a reversible connection fitting for attaching a second screw to the inner surface at the center of the inner surface of the top portion of the container body, and an O-ring reversibly connecting the second screw to the second end of the rod; The impeller assembly described therein is partially retractable; and The bioprocess container is configured to activate and expand T cells.

31. The bioprocess container of claim 30, wherein the shaft includes at least one hole, and the lever is capable of being fitted into the at least one hole.

32. The bioprocess container of claim 31, wherein rotating the lever in the at least one hole in a first direction will disengage the first screw from the second screw, thereby extending the impeller assembly, and rotating the lever in a second direction will engage the first screw with the second screw, thereby retracting the impeller assembly.

33. The bioprocess container of claim 30, wherein the impeller assembly is configured to retract from its fully extended position to a position between approximately one-sixth and approximately two-thirds of the height of the container body.

34. The bioprocess container of claim 33, wherein the impeller assembly is configured to retract from a fully extended position to a position between approximately one-third and approximately half the height of the container body.

35. The bioprocess container of claim 30, wherein the impeller assembly further comprises a magnet.

36. The bioprocess container of claim 35, wherein when the impeller assembly is in its maximum retracted state, it does not cause magnetic interference with the magnetic beads.

37. The bioprocess container of claim 30, wherein the first O-ring is connected to the first end of the rod, and the first end of the rod is located within an inner channel of the shaft of the impeller assembly.

38. The bioprocess container of claim 37, wherein the impeller assembly is rotatable against the rod.

39. A method for activating and expanding T cells, the method comprising the following steps: (a) Providing a bioprocess container according to any one of the preceding claims; (b) Providing the bioprocess vessel with an aqueous solution suitable for T cell activation and expansion; (c) Providing beads to the bioprocess container, wherein the beads contain a T-cell activator; (d) Providing T cells to the bioprocessing vessel; and (e) T cells activated by the beads are cultured in the bioprocessing container.

40. The method of claim 39, wherein steps (a) and (b) are performed before step (c), and wherein step (d) is performed after step (c).

41. The method of claim 39, wherein steps (a) and (b) are performed before step (d), and wherein step (c) is performed after step (d).

42. The method of claim 39, further comprising the step (f) of mixing the aqueous solution using the impeller of the impeller assembly.

43. The method of claim 39, wherein the impeller comprises a magnet located in the lower portion of the impeller.

44. The method of claim 39, wherein the T cells provided to the bioprocessing container are engineered T cells.

45. The method of claim 44, wherein the engineered T cell comprises a gene for a chimeric antigen receptor or a T cell receptor.

46. ​​The method of claim 43, wherein the bead is a magnetic bead, and wherein the magnetic bead is non-magnetically attached to the impeller magnet during activation and amplification.

47. The method of claim 39, wherein the T cell activator is an antigen of the αβ-T cell receptor on the T cell.

48. The method of claim 47, wherein the antigen comprises anti-CD3 or anti-CD28, or a combination thereof.

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