Cell selection and / or cell stimulation devices and methods of use

Abstract

Provided herein are devices and methods for selecting and stimulating a plurality of cells in a cell sample using column chromatography. In some aspects, the device includes a temperature control element for applying heat to a chromatographic stationary phase and a connector configured to provide air to the stationary phase during column chromatography. In some aspects, the devices and methods provided herein reduce the time required to produce a population of selected and stimulated cells useful for genetic modification, and thus for cell therapy, compared to existing devices and methods. TIFF2023500318000027.tif181137

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. US 62 / 928,303, filed October 30, 2019, entitled "CELL SELECTION AND / OR STIMULATION DEVICES AND METHODS OF USE," the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing. The Sequence Listing is provided in a file named 735042020040SeqList.txt, created on October 28, 2020, and is 74,908 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates to cell selection and / or cell stimulation devices and methods of use. In some aspects, the selected and / or stimulated cells are useful for genetic modification and thus for cell therapy. [Background technology]

[0004] background A variety of cell therapy approaches are available for treating diseases and conditions. Cell therapy approaches include those using immune cells, such as T cells (e.g., CD4+ T cells and CD8+ T cells), which may be genetically modified with recombinant receptors, such as chimeric antigen receptors. Improved devices and methods are needed to generate cell populations suitable for use in cell therapy and other applications. Devices, articles of manufacture, and methods are provided that meet these needs. Summary of the Invention

[0005] overview In some embodiments, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member and an outlet housing member, wherein at least the inlet housing member and the outlet housing member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving uptake of gas into the internal cavity, e.g., during at least a portion of a chromatography run. In some embodiments, the housing assembly further comprises a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form the internal cavity.

[0006] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving uptake of gas into the internal cavity.

[0007] In some embodiments, provided herein is a housing assembly for column chromatography that includes: a chromatography column including an internal cavity configured to accommodate a stationary phase; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving uptake of gas into the internal cavity. In some embodiments, the chromatography column includes an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form the internal cavity.

[0008] In any of the above embodiments, the connector may be disposed on the inlet housing member, the outlet housing member and / or the sidewall member.

[0009] In any of the above embodiments, the connector may be formed between any two or all three of the inlet housing member, the outlet housing member, and the sidewall member.

[0010] In any of the above aspects, the housing assembly may include a plurality of connectors.

[0011] In any of the above embodiments, the connector can be a bonded connector, a threaded connector, a luer connector (e.g., a luer lock connector or a luer slip connector), a barbed connector, or any combination thereof. In any of the above embodiments, the connector can be a luer lock connector or a luer slip connector. In any of the above embodiments, the connector can include a male fitting or a female fitting. In any of the above embodiments, the connector can be configured to sealingly engage tubing in fluid communication with the gas source. In any of the above embodiments, the connector can include one or more valves. In any of the above embodiments, the connector can be operably connected to tubing including one or more valves.

[0012] In any of the preceding embodiments, the connector can include one or more filters. In any of the preceding embodiments, the connector can be operatively connected to a tubing system that includes one or more filters. In any of the preceding embodiments, the one or more filters can be gas filters, e.g., air filters. In any of the preceding embodiments, the one or more filters can be air filters. In any of the preceding embodiments, the one or more filters can be sterile filters and / or sterilizing filters for sterile filtration. In any of the preceding embodiments, the one or more filters can be sterile filters. In any of the preceding embodiments, the one or more filters can be sterilizing filters for sterile filtration.

[0013] In any of the above aspects, the inlet housing member can include a top cover for the housing assembly. In some aspects, the top cover is removably attached to the inlet housing member or the sidewall member. In some aspects, the top cover is integrally formed with the inlet housing member or the sidewall member. In any of the above aspects, the connector can be disposed on the top cover.

[0014] In any of the foregoing embodiments, the inlet housing member can include one or more inlets operatively connected to the internal cavity to allow for the introduction of an input composition into the internal cavity. In some embodiments, the one or more inlets are located on the top cover. In some embodiments, the connector and the one or more inlets are located in the same or different locations on the top cover.

[0015] In any of the above embodiments, the fluid path through the one or more inlets can be at an angle of about 90 degrees relative to the top cover, while the fluid path through the connector can be at an angle of about 45 degrees relative to the top cover.

[0016] In any of the above aspects, the outlet housing member can include a bottom cover for the housing assembly. In some aspects, the bottom cover is removably attached to the outlet housing member or sidewall member, or the bottom cover is integrally formed with the outlet housing member or sidewall member.

[0017] In any of the above embodiments, the outlet housing member can include one or more outlets operatively connected to the internal cavity to enable or effect discharge of the output composition from the internal cavity. In some embodiments, the one or more outlets are disposed in the bottom cover. In some embodiments, the connector and the one or more outlets are disposed in the same or different locations on the bottom cover. In some embodiments, the fluid path through the one or more outlets is at an angle of about 90 degrees relative to the bottom cover.

[0018] In any of the above embodiments, the gas source can be or include a gas reservoir or the external environment. In any of the above embodiments, the gas in the gas source can be sterile. In any of the above embodiments, the gas can be or include air.

[0019] In any of the preceding embodiments, the housing assembly can further include tubing operatively connected to a gas source. In some embodiments, the tubing is configured to sterilely connect the interior cavity to the gas source. In any of the preceding embodiments, the tubing can include one or more valves. In any of the preceding embodiments, the tubing can include one or more filters.

[0020] In any of the foregoing embodiments, the housing assembly can further include one or more porous members, such as a cell strainer or cell sieve. In some embodiments, the one or more porous members can be cell strainers or cell sieves. In some embodiments, the housing assembly includes a first porous member configured to separate the stationary phase from an inlet of the internal cavity, the first porous member optionally being between the inlet housing member and the sidewall member. In some embodiments, the housing assembly further includes a second porous member configured to separate the stationary phase from an outlet of the internal cavity, the second porous member optionally being between the outlet housing member and the sidewall member.

[0021] In any of the preceding embodiments, the housing assembly can include: a first porous member configured to separate the stationary phase from an inlet of the internal cavity, optionally between the inlet housing member and the sidewall member; and / or a second porous member configured to separate the stationary phase from an outlet of the internal cavity, optionally between the outlet housing member and the sidewall member. In some embodiments, the first porous member or the second porous member is independently a cell strainer or cell sieve.

[0022] In any of the above embodiments, the first porous member can be between the inlet housing member and the sidewall member. In any of the above embodiments, the second porous member can be between the outlet housing member and the sidewall member.

[0023] In any of the above embodiments, the one or more porous members may have an average pore size of about 20 μm, or the one or more porous members may comprise a mesh having a mesh size of about 20 μm.

[0024] In any of the above embodiments, the temperature control member can be configured to adjust or maintain the temperature of the stationary phase in the internal cavity. In any of the above embodiments, the temperature control member can be configured to heat the stationary phase in the internal cavity from a starting temperature (e.g., room temperature) to a target temperature of about 35°C to about 39°C (e.g., 37°C or about 37°C). In any of the above embodiments, the target temperature can be 37°C or about 37°C. In some embodiments, the temperature control member is further configured to maintain the stationary phase at the target temperature.

[0025] In any of the above embodiments, the temperature control member can be configured to heat the stationary phase to a target temperature of about 30° C. to about 39° C. In any of the above embodiments, the target temperature can be about 35° C. to about 39° C., optionally 37° C. or about 37° C. In any of the above embodiments, the target temperature can be 37° C. or about 37° C. In some embodiments, the temperature control member is further configured to maintain the stationary phase at the target temperature.

[0026] In any of the above aspects, the housing assembly can include a temperature sensor configured to measure the temperature of the stationary phase in the internal cavity. The temperature sensor can form part of the temperature control member or can be separate from the temperature control member. In some aspects, the temperature sensor is configured to couple to a monitoring / display unit.

[0027] In any of the preceding aspects, the temperature control member can include a heat source. In any of the preceding aspects, the temperature control member can be configured to operatively connect to a heat source external to the housing assembly.

[0028] In any of the above aspects, the temperature control member can include a heating element or multiple heating elements.

[0029] In any of the above embodiments, the heating element and / or heating elements can be configured to heat the stationary phase evenly.

[0030] In any of the above embodiments, the temperature control member can comprise a heating element selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In any of the above embodiments, the temperature control member can comprise a plurality of heating elements, each selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In some embodiments, the heating element is an electric heating element. In some embodiments, at least one of the plurality of heating elements is an electric heating element. In some embodiments, the electric heating element comprises a metal plate, a metal rod, a metal wire, or a combination thereof. In some embodiments, the electric heating element can be configured to connect to a power source external to the housing assembly. In some embodiments, the heating element is an electromagnetic induction heating element. In some embodiments, at least one of the plurality of heating elements is an electromagnetic induction heating element. In some embodiments, the electromagnetic induction heating element comprises an induction heating coil surrounding a magnetizable core configured to provide heat to a stationary phase in the internal cavity. In some embodiments, the heating element is a non-electric heating element. In some embodiments, at least one of the plurality of heating elements is a non-electric heating element. In some embodiments, the non-electrical heating element includes a heating flow path including an inlet and an outlet for a heated fluid, such as a heated liquid or gas. In some embodiments, the heated fluid is a heated liquid or a heated gas. In some embodiments, the heating flow path can be a heating coil. In some embodiments, the heated fluid can be heated water. In some embodiments, the heating flow path is a heating coil and the heated fluid is heated water. In some embodiments, the heated water inlet is configured to connect to an external reservoir of heated water.

[0031] In any of the above embodiments, the heating element can be disposed along and / or around the central axis of the internal cavity. In any of the above embodiments, the heating element can be disposed inside the internal cavity, outside the internal cavity, or partially inside and partially outside the internal cavity. In any of the above embodiments, the heating element can be disposed inside the sidewall member, outside the sidewall member, or partially inside and partially outside the sidewall member. In any of the above embodiments, the heating element can include a coil surrounding the inlet housing member, the outlet housing member, and / or the sidewall member.

[0032] In any of the preceding embodiments, the heating element can include a heating channel surrounding the inlet housing member, the outlet housing member, and / or the sidewall member. In any of the preceding embodiments, the heating element can include a heating coil surrounding the inlet housing member, the outlet housing member, and / or the sidewall member. In any of the preceding embodiments, at least one of the multiple heating elements can be disposed along and / or around a central axis of the internal cavity. In any of the preceding embodiments, at least one of the multiple heating elements can be disposed inside the internal cavity, outside the internal cavity, or partially inside and partially outside the internal cavity. In any of the preceding embodiments, at least one of the multiple heating elements can be disposed inside the sidewall member, outside the sidewall member, or partially inside and partially outside the sidewall member.

[0033] In any of the preceding embodiments, the heating element and / or at least one of the plurality of heating elements can surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In any of the preceding embodiments, the heating element and / or at least one of the plurality of heating elements can surround at least a portion of the sidewall member.

[0034] In any of the above aspects, the plurality of heating elements can be evenly or approximately evenly distributed around the periphery of the sidewall member.

[0035] In any of the preceding embodiments, at least a portion of the heating element and / or at least a portion of at least one of the plurality of heating elements can be in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member, optionally at least a portion of the sidewall member. In any of the preceding embodiments, at least a portion of the heating element and / or at least a portion of at least one of the plurality of heating elements can be in contact with at least a portion of the sidewall member.

[0036] In any of the above embodiments, at least a portion of the heating element and / or at least a portion of at least one of the plurality of heating elements can be out of contact with the inlet housing member, the outlet housing member, or the sidewall member.

[0037] In any of the above embodiments, the housing assembly can further include an insulating layer between the heating element and / or at least one of the plurality of heating elements and at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the insulating layer can include a gas, optionally air, or a liquid. In some embodiments, the insulating layer can include air.

[0038] In any of the foregoing embodiments, the heating element can include a heating channel, and the heating channel can surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0039] In any of the foregoing embodiments, the heating element can include a heating coil, and the heating coil can surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0040] In any of the above embodiments, the plurality of heating elements can include a plurality of heating channels surrounding at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In any of the above embodiments, at least two of the plurality of heating channels can be fluidly coupled to each other.

[0041] In any of the above embodiments, the plurality of heating elements can be a plurality of electric heating elements surrounding at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In any of the above embodiments, at least two of the plurality of electric heating elements can be electrically coupled to one another. In any of the above embodiments, at least one of the plurality of electric heating elements can be configured to electrically connect to a power source external to the housing assembly.

[0042] In any of the preceding aspects, the housing assembly can further include a jacket member including a heating element or at least one of a plurality of heating elements, where the jacket member is configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In any of the preceding aspects, the housing assembly can further include a jacket member including a temperature control member including a heating element or at least one of a plurality of heating elements, where the jacket member is configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0043] In some embodiments, the jacket member surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0044] In any of the above embodiments, the jacket members can be removably connected together to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0045] In any of the preceding aspects, the jacket member can be configured to surround at least a portion of the sidewall member. Optionally, the jacket member can be configured to completely surround the sidewall member. In any of the preceding aspects, the jacket member can be configured to completely surround the sidewall member. In any of the preceding aspects, the jacket member can surround at least a portion of the sidewall member. Optionally, the jacket member can completely surround the sidewall member. In any of the preceding aspects, the jacket member can be configured to completely surround the sidewall member. In any of the preceding aspects, the jacket member can completely surround the sidewall member.

[0046] In any of the above embodiments, the jacket member can include two or more jacket parts that are together configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member, optionally completely surround the sidewall member. In any of the above embodiments, the jacket member can include two or more jacket parts that are together configured to surround at least a portion of the sidewall member. In any of the above embodiments, the jacket member can include two or more jacket parts that are together configured to completely surround the sidewall member.

[0047] In any of the above embodiments, a portion of the one or more inlets of the inlet housing member and / or a portion of the one or more outlets of the outlet housing member can be exposed by the jacket member. In any of the above embodiments, a portion of the one or more inlets of the inlet housing member and / or a portion of the one or more outlets of the outlet housing member can be outside the jacket member.

[0048] In any of the preceding embodiments, at least a portion of the jacket member can be in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member, optionally at least a portion of the sidewall member. In any of the preceding embodiments, at least a portion of the jacket member can be in contact with at least a portion of the sidewall member.

[0049] In any of the above embodiments, at least a portion of the jacket member can be out of contact with the inlet housing member, the outlet housing member, or the sidewall member.

[0050] In any of the foregoing embodiments, the heating element or at least one of the plurality of heating elements can be a heating channel including an inlet and an outlet for a heated fluid, and the jacket member can include at least one opening for the inlet for the heating fluid and at least one opening for the outlet for the heated fluid.

[0051] In any of the above embodiments, the heating element or at least one of the plurality of heating elements can be an electric heating element, and the jacket member can be arranged such that the electric heating element is configured to electrically connect to a power source external to the housing assembly.

[0052] In any of the above embodiments, the two or more jacket components can be configured to be removably connected together.

[0053] In any of the above embodiments, the jacket member can include a plurality of heating elements, and at least two of the two or more jacket components can each include at least one of the plurality of heating elements. In any of the above embodiments, at least two of the two or more jacket components can further include a temperature sensor.

[0054] In any of the preceding embodiments, at least two of the two or more jacket components can each include a heating flow path including an inlet and an outlet for a heated fluid, optionally heated water. In some embodiments, the heated fluid can be heated water. In any of the preceding embodiments, at least two of the heating flow paths of the two or more jacket components can be fluidly connected to one another. In any of the preceding embodiments, at least one inlet of at least two of the heating flow paths of the two or more jacket components can be configured to connect to an external reservoir of heated water.

[0055] In any of the above embodiments, at least two of the two or more jacket components can each include an electric heating element, optionally including a metal plate. In any of the above embodiments, the electric heating elements of at least two of the two or more jacket components can be electrically coupled to one another. In any of the above embodiments, the electric heating elements of at least two of the two or more jacket components can be configured to electrically connect to a power source external to the housing assembly.

[0056] In some embodiments, provided herein is a jacket member for column chromatography comprising one or more jacket components configured to surround at least a portion of a chromatography column and one or more heating elements, wherein the one or more heating elements are configured to provide heat to the chromatography column in the one or more jacket components.

[0057] In some embodiments, the one or more heating elements are configured to be part of a temperature control member, which is configured to regulate or maintain the temperature of the chromatography column.

[0058] In some embodiments, provided herein is a jacket element for column chromatography comprising one or more jacket components configured to surround at least a portion of a chromatography column and a temperature control member comprising one or more heating elements, wherein the one or more heating elements are configured to provide heat to a stationary phase in the one or more jacket components, and the temperature control member is configured to regulate or maintain a temperature of the chromatography column.

[0059] In some embodiments, one or more jacket components can be configured to be removably connected together and surround at least a portion of the chromatography column.

[0060] In any of the preceding aspects, the chromatography column is configured to contain a stationary phase.

[0061] In any of the above embodiments, the temperature control component can be configured to heat the chromatography column and / or stationary phase to a target temperature of about 30° C. to about 39° C. In any of the above embodiments, the target temperature can be about 35° C. to about 39° C., optionally 37° C. or about 37° C. In any of the above embodiments, the target temperature can be 37° C. or about 37° C.

[0062] In any of the above embodiments, the temperature control member can be further configured to maintain the chromatography column and / or stationary phase at a target temperature.

[0063] In any of the preceding embodiments, the jacket member can further include a temperature sensor configured to measure the temperature of the chromatography column and / or the stationary phase. In any of the preceding embodiments, one or more jacket components can further include a temperature sensor configured to measure the temperature of the chromatography column and / or the stationary phase.

[0064] In any of the preceding aspects, the temperature control member can include a heat source. In any of the preceding aspects, the temperature control member can be configured to be operatively connected to a heat source external to the jacket member.

[0065] In any of the above embodiments, at least a portion of the one or more heating elements and / or jacket member can be configured to be in contact with at least a portion of the chromatography column. In any of the above embodiments, at least a portion of the one or more heating elements and / or jacket member can be configured to be out of contact with at least a portion of the chromatography column.

[0066] In any of the above embodiments, the jacket member can be configured to allow for placement of an insulating layer between one or more jacket components and at least a portion of the chromatography column.

[0067] In any of the above embodiments, the jacket member can further include an insulating layer that can be configured to be disposed between one or more jacket components and at least a portion of the chromatography column.

[0068] In any of the above embodiments, the one or more heating elements can be configured to heat the stationary phase evenly in the one or more jacket components.

[0069] In any of the preceding embodiments, each of the one or more jacket components includes at least one of the one or more heating elements.

[0070] In any of the above embodiments, the one or more heating elements can each be selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In any of the above embodiments, the one or more heating elements can include a heating flow path including an inlet and an outlet for a heated fluid, and optionally, the heated fluid can be heated water. In any of the above embodiments, the heated fluid can be heated water. In some embodiments, the inlet can be configured to connect to an external reservoir of heated fluid.

[0071] In any of the preceding embodiments, the jacket member can include at least one opening for an inlet for a heated fluid. In any of the preceding embodiments, the jacket member can include at least one opening for an outlet for a heated fluid.

[0072] In any of the preceding embodiments, the one or more heating elements can include an electric heating element, and optionally, the electric heating element includes a metal plate. In some embodiments, the electric heating element includes a metal plate. In any of the preceding embodiments, the electric heating element can be configured to electrically connect to a power source external to the jacket member.

[0073] In any of the above embodiments, the jacket member can include two or more heating elements. In any of the above embodiments, the two or more heating elements can be configured to be evenly or nearly evenly distributed around the circumference of the chromatography column in one or more jacket components.

[0074] In any of the above embodiments, the temperature control member can include two or more heating channels, each including an inlet and an outlet for a heated fluid, and optionally, the heated fluid is heated water. In some embodiments, the two or more heating channels can be configured to be fluidly connected to one another.

[0075] In any of the above embodiments, the jacket member can include two or more electric heating elements, and optionally, the two or more electric heating elements include metal plates. In any of the above embodiments, the jacket member can include two or more electric heating elements including metal plates. In any of the above embodiments, the two or more electric heating elements can be configured to be electrically coupled to one another. In any of the above embodiments, at least one of the two or more electric heating elements can be configured to be electrically connected to a power source external to the jacket member.

[0076] In any of the preceding embodiments, the one or more jacket components can include two or more jacket components. In any of the preceding embodiments, the one or more jacket components can include two jacket components. In any of the preceding embodiments, the one or more jacket components can include three jacket components. In any of the preceding embodiments, the one or more jacket components can include four jacket components.

[0077] In any of the preceding embodiments, the two or more jacket components can be configured to be removably connected together and surround at least a portion of the chromatography column. In any of the preceding embodiments, at least two of the two or more jacket components can each include a heating element. In any of the preceding embodiments, at least two of the two or more jacket components can each further include a temperature sensor.

[0078] In any of the preceding embodiments, at least two of the two or more jacket components each include a heating flow path including an inlet and an outlet for a heated fluid, optionally for heated water. In any of the preceding embodiments, at least two of the two or more jacket components each include a heating flow path including an inlet and an outlet for heated water. In any of the preceding embodiments, at least two of the heating flow paths of the two or more jacket components can be configured to be fluidly connected to one another. In any of the preceding embodiments, at least one inlet of at least two of the heating flow paths of the two or more jacket components can be configured to connect to an external reservoir of heated water.

[0079] In any of the preceding embodiments, at least two of the two or more jacket components can each include an electric heating element, and optionally, the electric heating element includes a metal plate. In any of the preceding embodiments, at least two of the two or more jacket components can each include an electric heating element including a metal plate. In any of the preceding embodiments, the at least two electric heating elements of the two or more jacket components can be configured to be electrically coupled to each other. In any of the preceding embodiments, at least one of the at least two electric heating elements of the two or more jacket components can be configured to electrically connect to a power source external to the housing assembly.

[0080] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity and regulate or maintain a temperature of the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving uptake of gas into the internal cavity.

[0081] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element configured to provide heat to the stationary phase in the internal cavity and regulate or maintain the temperature of the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving the uptake of gas into the internal cavity.

[0082] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element disposed along and / or around a central axis of the internal cavity, the heating element configured to impart heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0083] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element comprising a metal plate configured to impart heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0084] In some embodiments, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element including a heating coil configured to impart heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of sterile gas into the internal cavity. In some embodiments, the heating coil includes an inlet and an outlet for heated water.

[0085] In any of the above embodiments, the heating coil may surround the inlet housing member, the outlet housing member, and the sidewall member.

[0086] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of a sterile gas into the internal cavity. In some embodiments, the gas filter is an air filter, and the sterile gas is sterile air. In any of the foregoing embodiments, the housing assembly may further comprise a gas filter.

[0087] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: a chromatography column including an internal cavity configured to accommodate a stationary phase; and a jacket member of any of the preceding embodiments configured to surround at least a portion of the chromatography column.

[0088] In some embodiments, the chromatography column can include an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an interior cavity. In some embodiments, the jacket member can be removably connected together and surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In any of the foregoing embodiments, the housing assembly can further include a connector configured to operably and sterilely connect the interior cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the interior cavity.

[0089] In some embodiments, provided herein is a housing assembly for column chromatography that includes: an inlet housing member, an outlet housing member, and a sidewall member that form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to regulate or maintain the temperature of the stationary phase, the temperature control member including a heating coil configured to apply heat to the stationary phase; a jacket member including the heating coil that is removably connected to surround at least a portion of the inlet housing member, the outlet housing member, and the sidewall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0090] In some embodiments, the heating coil completely surrounds the sidewall member. In some embodiments, the jacket member can include a second heating coil, and the heating coil and the second heating coil can collectively surround the sidewall member.

[0091] In some embodiments, provided herein is a housing assembly for column chromatography that includes: an inlet housing member, an outlet housing member, and a sidewall member that form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to regulate or maintain the temperature of the stationary phase, the temperature control member including a metal plate and an electric heating element configured to apply heat to the stationary phase; a jacket member including the electric heating element, the jacket member detachably connected to surround at least a portion of the inlet housing member, the outlet housing member, and the sidewall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0092] In some embodiments, the jacket member can include a plurality of electric heating elements, including metal plates, distributed evenly or approximately evenly around the periphery of the sidewall member.

[0093] In some embodiments, disclosed herein is a housing assembly set including a plurality of the housing assemblies of any of the preceding embodiments. In some embodiments, the housing assembly set includes at least two of the plurality of housing assemblies arranged in series. In any of the preceding embodiments, the housing assembly set can include at least two of the plurality of housing assemblies arranged in parallel.

[0094] In some embodiments, provided herein is a chromatography system comprising the housing assembly of any of the preceding embodiments and at least one additional chromatography column.

[0095] In some embodiments, provided herein is a chromatography kit including the housing assembly or housing assembly set of any of the preceding embodiments and a stationary phase for chromatography. In some embodiments, provided herein is a chromatography kit including the chromatography system of any of the preceding embodiments and a stationary phase for chromatography. In some embodiments, provided herein is a chromatography kit including the jacket member of any of the preceding embodiments, a chromatography column, and a stationary phase for chromatography.

[0096] In some embodiments, provided herein is a chromatography column or a chromatography column set comprising the housing assembly or housing assembly set of any of the preceding embodiments and a stationary phase for column chromatography in one or more internal cavities of the housing assembly. In some embodiments, provided herein is a chromatography column comprising the jacket member of any of the preceding embodiments and a chromatography column, wherein the internal cavity of the chromatography column comprises a stationary phase for chromatography. In some embodiments, provided herein is a chromatography column set comprising at least one jacket member of any of the preceding embodiments and a plurality of chromatography columns, wherein the internal cavity of each of the chromatography columns comprises a stationary phase for chromatography. In some embodiments, the plurality of chromatography columns are arranged in series or in parallel, and optionally, the plurality of chromatography columns are operatively connected. In some embodiments, the plurality of chromatography columns are operatively connected. In any of the preceding embodiments, the plurality of chromatographic columns can comprise a first chromatography column and a second chromatography column, and at least one jacket member can be configured to surround the second chromatography column. In some embodiments, the stationary phase comprises a gel filtration matrix. In any of the preceding embodiments, the stationary phase can comprise an affinity chromatography matrix. In any of the above aspects, the stationary phase may be or include a non-magnetic, non-ferromagnetic or paramagnetic material.In any of the above embodiments, the stationary phase may be or comprise a material selected from the group consisting of cellulose membranes, plastic membranes, polysaccharide gels, polyacrylamide gels, agarose gels, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, poly(N-isopropylacrylamide)-grafted silica, styrene-divinylbenzene gels, copolymers of acrylates or acrylamides and diols, copolymers of polysaccharides and N,N'-methylenebisacrylamide, and combinations thereof. In any of the above embodiments, the stationary phase may be or comprise a monolithic matrix, a particulate matrix, and / or a planar matrix. In some embodiments, the particulate matrix has an average particle size of about 5 μm to about 200 μm, about 5 μm to about 600 μm, or about 5 μm to about 1500 μm. In any of the above embodiments, the stationary phase may have an average pore size of about 1 nm to about 500 nm.

[0097] In any of the foregoing embodiments, the stationary phase may include a selection agent immobilized thereon. In some embodiments, the selection agent is capable of specifically binding to a selection marker on the surface of one or more cells. In any of the foregoing embodiments, the one or more cells may be or include immune cells. In some embodiments, the one or more cells are T cells.

[0098] In any of the preceding embodiments, the selection agent can be or can comprise an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof. In any of the preceding embodiments, the selection agent can be or can comprise an antibody fragment. In any of the preceding embodiments, the selection agent can be or can comprise a Fab fragment. In any of the preceding embodiments, the selection agent can be or can comprise a bivalent antibody fragment selected from the group of bivalent antibody fragments consisting of F(ab')2 fragments and bivalent single-chain Fv (scFv) fragments. In any of the preceding embodiments, the selection agent can be or can comprise a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFv. In any of the above aspects, the selection agent can be or comprise a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

[0099] In any of the preceding embodiments, the selection agent may be biotin, a biotin analogue that reversibly binds to streptavidin or avidin, TIFF0007746263000001.tif41145, a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag. In any of the foregoing embodiments, the selection agent can comprise a streptavidin-binding peptide.

[0100] In any of the preceding embodiments, the selection marker is or can comprise a T cell coreceptor. In any of the preceding embodiments, the selection marker is or can comprise a member of the T cell antigen receptor complex. In any of the preceding embodiments, the selection marker is or can comprise a CD3 complex. In any of the preceding embodiments, the selection marker is or can comprise a CD3 chain. In any of the preceding embodiments, the selection marker is or can comprise a CD3 chain. In any of the preceding embodiments, the selection marker is or can comprise a CD3γ, CD3δ, CD3ε, or CD3ζ chain. In any of the preceding embodiments, the selection marker is or can comprise CD8. In any of the preceding embodiments, the selection marker is or can comprise CD4. In any of the preceding embodiments, the selection marker is or can comprise CD45RA. In any of the preceding embodiments, the selection marker is or can comprise CD27. In any of the preceding embodiments, the selection marker is or can comprise CD28. In any of the above embodiments, the selectable marker can be or include CCR7.

[0101] In any of the above embodiments, specific binding between the selection agent and the selection marker may not result in the induction of a signal to the T cell, such as the induction of a stimulatory, activation or proliferation signal.

[0102] In any of the foregoing embodiments, the selection agent is or can comprise an anti-CD3 Fab, an anti-CD8 Fab, or an anti-CD4 Fab. In any of the foregoing embodiments, the selection agent is or can comprise an anti-CD27 Fab. In any of the foregoing embodiments, the selection agent can be directly or indirectly bound to the stationary phase. In any of the foregoing embodiments, the selection agent can be indirectly bound to the stationary phase via a selection reagent to which the selection agent reversibly binds.

[0103] In any of the foregoing embodiments, the selection reagent may be or may comprise streptavidin, avidin, or a mutein of streptavidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof; an avidin or mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K, wherein said at least two chelating groups are capable of binding to a transition metal ion; an agent capable of binding to an oligohistidine affinity tag; an agent capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding to calmodulin-binding peptide (CBP); an agent capable of binding to a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose-binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein. In any of the foregoing embodiments, the selection reagent can be or include a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide.

[0104] In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatographic columns can be an anti-CD4 antibody (e.g., an anti-CD4 Fab) and the selection substance immobilized on the stationary phase of at least one other of the plurality of chromatographic columns can be an anti-CD8 antibody (e.g., an anti-CD8 Fab). In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatographic columns can be an anti-CD4 Fab and the selection substance immobilized on the stationary phase of at least one other of the plurality of chromatographic columns can be an anti-CD8 Fab.

[0105] In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatographic columns can be an anti-CD3 antibody (e.g., an anti-CD3 Fab), and the selection substance immobilized on the stationary phase of at least one other chromatographic column of the plurality of chromatographic columns can be an antibody targeting CD45RA, CD27, CD28, or CCR7, optionally an anti-CD27 antibody (e.g., an anti-CD27 Fab). In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatographic columns can be an anti-CD3 Fab, and the selection substance immobilized on the stationary phase of at least one other chromatographic column of the plurality of chromatographic columns can be an anti-CD27 Fab.

[0106] In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD4 antibody (e.g., an anti-CD4 Fab), the selection substance immobilized on at least one other chromatography column of the plurality of chromatography columns can be an anti-CD8 antibody (e.g., an anti-CD8 Fab), and the selection substance immobilized on the stationary phase of at least one further chromatography column of the plurality of chromatography columns can be an antibody targeting CD45RA, CD27, CD28, or CCR7, optionally an anti-CD27 antibody (e.g., an anti-CD27 Fab). In any of the foregoing embodiments, the selection substance immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD4 Fab, the selection substance immobilized on at least one other chromatography column of the plurality of chromatography columns can be an anti-CD8 Fab, and the selection substance immobilized on the stationary phase of at least one further chromatography column of the plurality of chromatography columns can be an anti-CD27 Fab.

[0107] In any of the above embodiments, the chromatography kit, chromatography column, or chromatography column set may further comprise one or more stimulatory substances capable of delivering a stimulatory signal to one or more T cells. In some embodiments, the stationary phase comprises at least one of the one or more stimulatory substances. In some embodiments, the one or more stimulatory substances are immobilized on the stationary phase of the chromatography column or chromatography column set. In some embodiments, the one or more stimulatory substances are indirectly immobilized. In some embodiments, the one or more stimulatory substances are indirectly immobilized via a streptavidin mutein that reversibly binds to a streptavidin-binding peptide. In some embodiments, the chromatography kit may comprise a stimulatory reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal to one or more T cells. In some embodiments, the at least one stimulatory substance or at least one of the one or more stimulatory substances is a first stimulatory substance, and the chromatography kit, chromatography column, or chromatography column set further comprises one or more second stimulatory substances capable of enhancing, attenuating, or modifying the stimulatory signal of the first stimulatory substance. In some embodiments, at least one of the second stimulatory agents is capable of specifically binding to one or more costimulatory molecules on T cells, such as CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In any of the foregoing embodiments, the stationary phase can comprise at least one of the one or more second stimulatory agents.

[0108] In any of the above embodiments, the stimulatory signal can be a signal from the TCR / CD3 complex in the T cell, a CD3-containing complex in the T cell, and / or an ITAM-containing molecule in the T cell.

[0109] In any of the foregoing embodiments, the one or more stimuli can be or can comprise an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof. In any of the foregoing embodiments, the one or more stimuli can be or can comprise an antibody fragment. In any of the foregoing embodiments, the one or more stimuli can be or can comprise a Fab fragment. In any of the foregoing embodiments, the one or more stimuli can be or can comprise a bivalent antibody fragment selected from the group of bivalent antibody fragments consisting of an F(ab')2 fragment and a bivalent single-chain Fv (scFv) fragment. In any of the foregoing embodiments, the one or more stimuli can be or can comprise a monovalent antibody fragment selected from the group consisting of an Fab fragment, an Fv fragment, and an scFv. In any of the above embodiments, the one or more stimulatory agents can be or include a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

[0110] In any of the preceding embodiments, the one or more stimulatory agents may be biotin, a biotin analog that reversibly binds to streptavidin or avidin, The peptide may further comprise a streptavidin-binding peptide selected from the group consisting of: TIFF0007746263000002.tif41140; a calmodulin-binding peptide that reversibly binds to calmodulin; a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide; and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.

[0111] In any of the preceding embodiments, the first and second stimulating substances can independently be or comprise an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof. In any of the preceding embodiments, the first and second stimulating substances can independently be or comprise an antibody fragment. In any of the preceding embodiments, the first and second stimulating substances can independently be or comprise a Fab fragment. In any of the preceding embodiments, the first and second stimulating substances can independently be or comprise a bivalent antibody fragment selected from the group of bivalent antibody fragments consisting of an F(ab')2 fragment and a bivalent single-chain Fv (scFv) fragment. In any of the preceding embodiments, the first and second stimulating substances can independently be or comprise a monovalent antibody fragment selected from the group consisting of an Fab fragment, an Fv fragment, and an scFv. In any of the foregoing embodiments, the first and second stimulatory agents can independently be or include proteinaceous binding molecules with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers. In some embodiments, the first stimulatory reagent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab.

[0112] In any of the preceding embodiments, the first and second stimulatory agents are independently biotin, streptavidin, or a biotin analog that reversibly binds to avidin; TIFF0007746263000003.tif47140, a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag. In any of the foregoing embodiments, the first and second stimulatory substances can independently further comprise a streptavidin-binding peptide.

[0113] In any of the preceding embodiments, the streptavidin-binding peptide is TIFF0007746263000004.tif41142.

[0114] In any of the foregoing embodiments, the first stimulant and the second stimulant can reversibly bind to an oligomeric stimulant reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the size of the oligomeric stimulant reagent is: i) greater than 50 nm in radius; ii) at least 5×10 6 g / mol molecular weight, and / or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulation reagent.

[0115] In any of the above embodiments, the stimulating reagent can comprise a plurality of streptavidin or streptavidin mutein molecules, wherein the size of the stimulating reagent is: i) greater than 50 nm in radius; ii) at least 5×10 6 g / mol molecular weight, and / or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per stimulation reagent.

[0116] In any of the foregoing embodiments, the streptavidin mutein comprises the amino acid sequence Val(III) at sequence positions corresponding to positions 44-47 of SEQ ID NO:1. 44 -Thr 45 -Ala46 -Arg 47 or the streptavidin mutein may comprise the amino acid sequence Ile at a sequence position corresponding to positions 44-47 of SEQ ID NO:1. 44 -Gly 45 -Ala 46 -Arg 47 In any of the foregoing embodiments, the N-terminal amino acid residue of the streptavidin mutein can be within the region of amino acids 10 to 16 of SEQ ID NO: 1, and the C-terminal amino acid residue of the streptavidin mutein can be within the region of amino acids 133 to 142 of SEQ ID NO: 1. In any of the foregoing embodiments, the streptavidin mutein can comprise the amino acid sequence set forth in any of SEQ ID NOs: 3 to 6, 27, 28, 104, and 105.

[0117] In some embodiments, disclosed herein is a device comprising the housing assembly, housing assembly set, or chromatography kit, chromatography column, or chromatography column set of any of the preceding embodiments, wherein the device further comprises an input composition reservoir operably connected to the internal cavity via an inlet of the inlet housing member. In some embodiments, disclosed herein is a device comprising the chromatography system of any of the preceding embodiments, wherein the device further comprises an input composition reservoir operably connected to the internal cavity via an inlet of the inlet housing member. In some embodiments, disclosed herein is a device comprising the jacket member of any of the preceding embodiments and a chromatography column or chromatography column set, wherein the chromatography column comprises an internal cavity configured to accommodate a stationary phase for chromatography, and the device further comprises an input composition reservoir operably connected to an inlet of the internal cavity to allow uptake of the input composition contained in the input composition reservoir into the internal cavity. In some embodiments, the input composition comprises blood or a blood-derived sample, or is blood or a blood-derived sample. In some embodiments, the input composition comprises or is a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. In some embodiments, the apheresis product or leukocyte apheresis product is freshly isolated from a subject or thawed from a frozen apheresis product or leukocyte apheresis product.

[0118] In any of the foregoing embodiments, the device can further include an output composition reservoir operatively connected to the internal cavity via an outlet in the outlet housing member. In any of the foregoing embodiments, the device can further include an output composition reservoir operatively connected to an outlet of the internal cavity to enable or effect discharge of the output composition contained in the output composition reservoir from the internal cavity. In some embodiments, the output composition includes or is enriched T cells. In some embodiments, the enriched T cells have been stimulated during chromatography on a chromatography column. In any of the foregoing embodiments, the device can be in a closed system or a sterile system.

[0119] In some embodiments, disclosed herein are methods of preparing a chromatography column or a set of chromatography columns, comprising introducing a stationary phase into a housing assembly or a set of housing assemblies of any of the preceding embodiments.

[0120] In some embodiments, disclosed herein are methods for preparing a chromatography column or a chromatography column set, comprising introducing the stationary phase of the chromatography kit of any of the preceding embodiments into a housing assembly or housing assembly set of the chromatography kit.

[0121] In some embodiments, disclosed herein are methods for on-column stimulation of T cells, comprising incubating a sample comprising a plurality of T cells on a chromatography column or set of chromatography columns of any of the preceding embodiments with one or more stimulatory substances to deliver a stimulatory signal to one or more T cells of the plurality of T cells immobilized on the stationary phase, thereby generating a composition comprising stimulated T cells as an output composition of the chromatography column or set of chromatography columns. In some embodiments, disclosed herein are methods for on-column stimulation of T cells, comprising incubating a sample comprising a plurality of T cells on a chromatography column or set of chromatography columns of any of the preceding embodiments with one or more stimulatory substances to deliver a stimulatory signal to one or more T cells of the plurality of T cells immobilized on the stationary phase, and collecting the one or more T cells from the stationary phase after the start of incubation, thereby generating an output composition comprising stimulated T cells. In some embodiments, the stationary phase comprises a selection substance that specifically binds to a selection marker on the surface of one or more T cells. In some embodiments, immobilization of the one or more T cells on the stationary phase is achieved by specific binding of the selection substance to a selection marker expressed by one or more T cells. In any of the foregoing embodiments, the stationary phase can include a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and immobilization of the one or more T cells in the stationary phase can be achieved by specific binding of the selection agent to the selection marker expressed by the one or more T cells. In any of the provided methods, during at least a portion of the incubation, the temperature control member regulates the temperature of the stationary phase to a target temperature that is higher than room temperature. In some embodiments, the target temperature is a physiological temperature that maximizes cellular health and activity in preparation for efficient or effective delivery of a stimulatory signal in the one or more T cells.

[0122] In any of the foregoing embodiments, the method may further comprise collecting one or more T cells from the stationary phase after the start of incubation. In some embodiments, the one or more T cells are collected from the stationary phase within 24 hours of the start of incubation. In any of the foregoing embodiments, the one or more T cells can be collected from the stationary phase within 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours of the start of incubation. In any of the foregoing embodiments, the one or more T cells can be collected from the stationary phase by gravity flow. In some embodiments, collection by gravity flow can comprise adding a wash medium to a chromatography column or housing assembly containing the stationary phase. In any of the foregoing embodiments, the collecting step can be performed without adding a competitor or free binding agent for eluting the plurality of T cells from the stationary phase. In any of the foregoing embodiments, the wash medium can be free of a competitor or free binding agent for eluting the one or more T cells from the stationary phase.

[0123] In some embodiments, the competitor or free binder can be an agent that competes with the streptavidin-binding peptide of the selection agent for binding to the streptavidin mutein immobilized on the stationary phase. In some embodiments, the competitor or free binder can be biotin or a biotin analog, optionally the biotin analog is D-biotin. In some embodiments, the competitor or free binder can be D-biotin.

[0124] In some embodiments, disclosed herein are methods of on-column stimulation of T cells, comprising: (a) adding a sample comprising a plurality of T cells to a stationary phase in a chromatography column or set of chromatography columns of any of the preceding embodiments, wherein the stationary phase comprises a selection agent that binds to a selection marker on the surface of one or more of said plurality of T cells, thereby immobilizing said one or more of said plurality of T cells on the stationary phase; and (b) adding to the stationary phase in the chromatography column or set of chromatography columns a stimulating reagent comprising one or more stimulating agents capable of delivering a stimulatory signal in one or more of said plurality of T cells, thereby initiating incubation of the stimulating reagent with said one or more T cells, thereby producing a composition comprising stimulated T cells as an output composition of the chromatography column or set of chromatography columns.

[0125] In some embodiments, disclosed herein are methods for on-column stimulation of T cells, comprising: (a) adding a sample comprising a plurality of T cells to an interior cavity of the chromatography column or set of chromatography columns of any of the preceding embodiments, the interior cavity comprising a stationary phase comprising a selection agent that binds to a selection marker on the surface of one or more of the plurality of T cells, thereby immobilizing one or more of the plurality of T cells on the stationary phase; (b) adding a stimulating reagent to the stationary phase in the chromatography column or set of chromatography columns, the stimulating reagent comprising one or more stimulating agents capable of delivering a stimulatory signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and (c) after the initiation of incubation, collecting the one or more T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.

[0126] In some embodiments, disclosed herein are methods for on-column stimulation of T cells, comprising: (a) mixing (i) a sample comprising a plurality of T cells with (ii) a stationary phase in the chromatography kit of any of the preceding embodiments, the stationary phase comprising a selection substance capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, wherein immobilization of the plurality of T cells on the stationary phase is achieved by specific binding of the selection substance to the selection marker; and (b) adding to the stationary phase a stimulation reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in the T cell, thereby initiating incubation of the stimulation reagent with the one or more T cells, wherein the mixing and / or adding steps are performed inside or outside the internal cavity of a chromatography column or set of chromatography columns of the chromatography kit, thereby producing a composition comprising stimulated T cells as an output composition of the chromatography column or set of chromatography columns.

[0127] In some embodiments, disclosed herein are methods for on-column stimulation of T cells, comprising: (a) mixing (i) a sample comprising a plurality of T cells with (ii) a stationary phase in the chromatography kit of any of the preceding embodiments, the stationary phase comprising a selection substance capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, wherein immobilization of the plurality of T cells on the stationary phase is achieved by specific binding of the selection substance to the selection marker; (b) adding to the stationary phase a stimulation reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in the T cell, thereby initiating incubation of the stimulation reagent with the one or more T cells, wherein the mixing and / or adding steps are performed inside or outside the internal cavity of a chromatography column or set of chromatography columns of the chromatography kit; and (c) after the initiation of incubation, collecting the one or more T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.

[0128] In any of the foregoing embodiments, the method may further comprise collecting one or more T cells from the stationary phase after the start of incubation. In some embodiments, the one or more T cells are collected from the stationary phase within 24 hours of the start of incubation. In any of the foregoing embodiments, the one or more T cells can be collected from the stationary phase within 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours of the start of incubation. In any of the foregoing embodiments, the one or more T cells can be collected from the stationary phase by gravity flow. In some embodiments, collection by gravity flow can include adding a wash medium to a chromatography column or housing assembly containing the stationary phase. In any of the foregoing embodiments, the collecting step can be performed without adding a competitor or free binding agent for eluting the plurality of T cells from the stationary phase. In any of the foregoing embodiments, the wash medium can be free of a competitor or free binding agent for eluting the one or more T cells from the stationary phase.

[0129] In some embodiments, the competitor or free binder can be an agent that competes with the streptavidin-binding peptide of the selection agent for binding to the streptavidin mutein immobilized on the stationary phase. In some embodiments, the competitor or free binder can be biotin or a biotin analog, optionally the biotin analog is D-biotin. In some embodiments, the competitor or free binder can be D-biotin.

[0130] In any of the foregoing embodiments, the stimulatory agent may be or include an oligomeric stimulatory reagent comprising: (i) a plurality of streptavidin or streptavidin mutein molecules; and (ii) one or more stimulatory agents capable of delivering a stimulatory signal in one or more T cells, wherein the size of the oligomeric stimulatory reagent is: i) greater than 50 nm in radius; ii) at least 5×10 6and / or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulation reagent. In some embodiments, the streptavidin mutein contains the amino acid sequence Val(III) at sequence positions corresponding to positions 44-47 in streptavidin in the sequence of amino acids set forth in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 or the streptavidin mutein comprises the amino acid sequence Val at a sequence position corresponding to positions 44 to 47 in streptavidin in the sequence of amino acids set forth in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47 Includes:

[0131] In any of the foregoing embodiments, at least one of the one or more stimulatory agents can be capable of delivering a stimulatory signal through the TCR / CD3 complex in a T cell, a CD3-containing complex in a T cell, and / or an ITAM-containing molecule in a T cell.

[0132] In any of the foregoing embodiments, at least one of the one or more stimulatory substances can be a first stimulatory substance capable of delivering a stimulatory signal, and the one or more stimulatory substances can further include one or more second stimulatory substances capable of enhancing, attenuating, or modifying the stimulatory signal of the first stimulatory substance. In some embodiments, the second stimulatory substance can be capable of specifically binding to a costimulatory molecule on the one or more T cells. In some embodiments, the costimulatory molecule can be selected from among CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In any of the foregoing embodiments, the second stimulatory substance can be capable of specifically binding to CD28, and / or the costimulatory molecule is CD28.

[0133] In any of the foregoing embodiments, the first stimulator can specifically bind to CD3, and the second stimulator can specifically bind to CD28. In any of the foregoing embodiments, the first stimulator can comprise a monovalent antibody fragment that binds to CD3, and the second stimulator can comprise a monovalent antibody fragment that binds to CD28. In some embodiments, the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In any of the foregoing embodiments, the first stimulator can be an anti-CD3 Fab, and the second stimulator can be an anti-CD28 Fab.

[0134] In any of the above embodiments, during at least a portion of the incubation, the temperature control member is capable of regulating the temperature of the stationary phase to a target temperature of about 30°C to about 39°C.

[0135] In any of the above embodiments, during at least a portion of the incubation, the temperature control member may regulate the temperature of the stationary phase to a target temperature of about 35°C to about 39°C.

[0136] In any of the above embodiments, the temperature control member is capable of maintaining the temperature of the stationary phase at a target temperature of about 30°C to about 39°C during at least a portion of the incubation.

[0137] In any of the above embodiments, the temperature control member is capable of maintaining the temperature of the stationary phase at a target temperature of about 35°C to about 39°C during at least a portion of the incubation.

[0138] In any of the above embodiments, the target temperature is about 30° C. to about 39° C., optionally 37° C. or about 37° C. In any of the above embodiments, the target temperature can be 37° C. or about 37° C.

[0139] In any of the preceding embodiments, the connector may allow introduction of gas into the internal cavity during at least a portion of the incubation. In some embodiments, the gas is sterile and is or comprises air. In any of the preceding embodiments, introduction of gas into the internal cavity may be intermittent or continuous during the incubation.

[0140] In any of the above aspects, the method may further comprise washing the stationary phase with medium, which medium does not contain a competitor or free binding agent for eluting the T cells from the stationary phase.

[0141] In any of the foregoing embodiments, the method may further comprise incubating the composition comprising the stimulated T cells. In some embodiments, the further incubation is carried out at or about 37°C ± 2°C; and / or the further incubation is carried out in the presence of an additional agent capable of delivering a signal to the T cells. In some embodiments, the additional agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In any of the foregoing embodiments, the additional agent is or can comprise a cytokine selected from among IL-2, IL-15, and IL-7. In any of the foregoing embodiments, the method may further comprise adding a competitor or free binder to the composition comprising the stimulated T cells. In any of the foregoing embodiments, the method may further comprise selecting and / or stimulating the stimulated T cells. In any of the foregoing embodiments, the method may further comprise introducing a recombinant nucleic acid molecule into the stimulated T cells of the composition, the nucleic acid molecule encoding a recombinant protein, thereby producing a composition comprising transduced T cells. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a chimeric antigen receptor. In some embodiments, a chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM. In some embodiments, the CAR further comprises a transmembrane domain that connects the extracellular domain and the intracellular signaling domain.

[0142] In any of the preceding aspects, the introduction of the recombinant nucleic acid can be achieved by transduction using a viral particle. In any of the preceding aspects, the introduction of the recombinant nucleic acid encoding the recombinant protein is by transduction using a viral particle.

[0143] In any of the foregoing embodiments, the recombinant nucleic acid can be introduced into the stimulated T cells before, during, or after incubation. In some embodiments, the recombinant nucleic acid is introduced into the stimulated T cells during incubation. In some embodiments, the recombinant nucleic acid is introduced into the stimulated T cells after incubation.

[0144] In any of the above embodiments, the method can further comprise adding a competitor or free binder to the composition comprising the transduced T cells.

[0145] In any of the preceding embodiments, the method can further comprise culturing the composition comprising the transduced cells under conditions for viral integration, thereby producing a composition comprising cultured T cells. In any of the preceding embodiments, the method can further comprise incubating the composition comprising the transduced cells under conditions for viral integration.

[0146] In any of the above aspects, the method can further comprise culturing the composition comprising the transduced cells under conditions for expanding the T cells.

[0147] In any of the above aspects, the method can further comprise culturing the composition comprising the transduced cells under conditions that do not substantially expand the T cells.

[0148] In any of the above embodiments, the method can further comprise adding a competitor or free binder to the composition comprising the cultured T cells.

[0149] In any of the foregoing embodiments, the method can further comprise formulating the cells of the output composition for cryopreservation and / or administration to a subject, optionally in the presence of a pharmaceutically acceptable excipient. In some embodiments, the cells of the output composition are formulated in the presence of a cryoprotectant.

[0150] In any of the above embodiments, at least one or all of the steps of the method may be performed in a closed system. In some embodiments, the closed system is automated.

[0151] In any of the foregoing embodiments, the sample may be or may comprise a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. In some embodiments, the apheresis product or leukocyte apheresis product is freshly isolated from a subject. In some embodiments, the apheresis product or leukocyte apheresis product is thawed from a frozen apheresis product or leukocyte apheresis product. [The present invention 1001] A housing assembly for column chromatography, comprising: an inlet housing member and an outlet housing member, at least the inlet housing member and the outlet housing member forming an interior cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and A connector configured to operatively connect the internal cavity to a gas source, thereby enabling or achieving the introduction of gas into the internal cavity. [The present invention 1002] The housing assembly of the present invention 1001 further comprising a sidewall member, wherein the inlet housing member, the outlet housing member and the sidewall member form an interior cavity. [The present invention 1003] The housing assembly of invention 1001 or invention 1002, wherein the connector includes one or more filters. [The present invention 1004] The housing assembly of the present invention 1003, wherein the one or more filters are gas filters. [The present invention 1005] The housing assembly of any of inventions 1001-1004, wherein the inlet housing member includes one or more inlets operatively connected to the internal cavity to allow intake of an input composition into the internal cavity. [The present invention 1006] The housing assembly of any of inventions 1001-1005, wherein the outlet housing member includes one or more outlets operatively connected to the internal cavity to enable or effect discharge of the output composition from the internal cavity. [The present invention 1007] The housing assembly of any one of 1001 to 1006, wherein the gas source is or includes a gas reservoir or the external environment. [The present invention 1008] The housing assembly of any one of 1001 to 1007, wherein the gas in the gas source is sterile. [The present invention 1009] The housing assembly of any one of 1001 to 1008, wherein the gas is air. [The present invention 1010] a first porous member configured to separate the stationary phase from the inlet of the internal cavity, optionally between the inlet housing member and the sidewall member; and / or a second porous member configured to separate the stationary phase from the outlet of the internal cavity, optionally between the outlet housing member and the sidewall member; The housing assembly of any one of 1001 to 1009 of the present invention further comprises: [The present invention 1011] The housing assembly of the present invention 1010, wherein the first porous member or the second porous member is independently a cell strainer or cell sieve. [The present invention 1012] The housing assembly of any one of claims 1001 to 1011, wherein the temperature control member is configured to regulate or maintain the temperature of the stationary phase in the internal cavity. [The present invention 1013] The housing assembly of any of inventions 1001 to 1012, wherein the temperature control member is configured to heat the stationary phase to a target temperature of about 30°C to about 39°C. [The present invention 1014] The housing assembly of the present invention 1013, wherein the target temperature is about 35°C to about 39°C, optionally 37°C or about 37°C. [The present invention 1015] The housing assembly of invention 1013 or invention 1014, wherein the temperature control member is further configured to maintain the stationary phase at a target temperature. [The present invention 1016] The housing assembly of any of claims 1001 to 1015, further comprising a temperature sensor configured to measure the temperature of the stationary phase in the internal cavity. [The present invention 1017] The housing assembly of any one of claims 1001 to 1016, wherein the temperature control member includes a heat source. [The present invention 1018] The housing assembly of any one of claims 1001 to 1016, wherein the temperature control member is configured to operatively connect to a heat source external to the housing assembly. [The present invention 1019] The housing assembly of any one of claims 1001 to 1018, wherein the temperature control member comprises a heating element or multiple heating elements. [The present invention 1020] The housing assembly of the present invention 1019, wherein the heating element and / or multiple heating elements are configured to heat the stationary phase evenly. [The present invention 1021] The housing assembly of any one of inventions 1001 to 1020, wherein the temperature control member comprises a heating element selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. [The present invention 1022] A housing assembly of any one of inventions 1001 to 1021, wherein the temperature control member includes a plurality of heating elements each selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. [The present invention 1023] A housing assembly according to any one of the present inventions 1019 to 1022, wherein at least one of the heating elements and / or a plurality of heating elements is an electric heating element. [The present invention 1024] A housing assembly according to the present invention 1023, wherein the electric heating element includes a metal plate, a metal rod, a metal wire, or a combination thereof. [The present invention 1025] A housing assembly according to the present invention 1023 or the present invention 1024, wherein the electric heating element is configured to be connected to a power source outside the housing assembly. [The present invention 1026] A housing assembly according to any one of the present inventions 1019 to 1025, wherein at least one of the heating elements and / or a plurality of heating elements is an electromagnetic induction heating element, and the electromagnetic induction heating element includes an induction heating coil surrounding a magnetizable core configured to heat a stationary phase in an internal cavity. [The present invention 1027] A housing assembly according to any one of the present inventions 1019 to 1026, wherein at least one of the heating elements and / or a plurality of heating elements is a non-electric heating element, and the non-electric heating element includes a heating flow path including an inlet and an outlet for a heated fluid. [The present invention 1028] A housing assembly according to the present invention 1027, wherein the heating flow path is a heating coil. [The present invention 1029] A housing assembly according to the present invention 1027 or the present invention 1028, wherein the heated fluid is heated water. [The present invention 1030] A housing assembly according to the present invention 1029, wherein the inlet for the heated water is configured to be connected to an external reservoir of the heated water. [The present invention 1031] A housing assembly according to any one of the present inventions 1019 to 1030, wherein at least one of the heating elements and / or a plurality of heating elements is arranged along the central axis of the internal cavity and / or around the central axis of the internal cavity. [The present invention 1032] A housing assembly according to any one of the present inventions 1019 to 1031, wherein at least one of the heating elements and / or a plurality of heating elements is arranged inside the internal cavity, outside the internal cavity, or partly inside and partly outside the internal cavity. [The present invention 1033] A housing assembly according to any one of the present inventions 1019 to 1032, wherein at least one of the heating elements and / or a plurality of heating elements is arranged inside the side wall member, outside the side wall member, or partly inside and partly outside the side wall member. [The present invention 1034] A housing assembly of any of inventions 1019 to 1033, wherein the heating element and / or at least one of the plurality of heating elements surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member. [This invention 1035] The housing assembly of any one of inventions 1019 to 1034, wherein the heating element and / or at least one of the plurality of heating elements surrounds at least a portion of the sidewall member. [The present invention 1036] The housing assembly of any one of inventions 1019 to 1035, wherein the plurality of heating elements are distributed evenly or approximately evenly around the outer periphery of the sidewall member. [This invention 1037] A housing assembly of any of inventions 1019 to 1036, wherein at least a portion of the heating element and / or at least a portion of at least one of the plurality of heating elements is in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member, optionally at least a portion of the side wall member. [The present invention 1038] The housing assembly of any of inventions 1034 to 1037, further comprising an insulating layer between the heating element and / or at least one of the plurality of heating elements and at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member. [This invention 1039] The housing assembly of the present invention 1038, wherein the insulating layer comprises a gas, optionally air, or a liquid. [The present invention 1040] A housing assembly of any of inventions 1019 to 1039, wherein the plurality of heating elements includes a plurality of heating channels surrounding at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member. [The present invention 1041] The housing assembly of the present invention 1040, wherein at least two of the plurality of heating channels are fluidly connected to each other. [The present invention 1042] A housing assembly of any of inventions 1019 to 1041, wherein the plurality of heating elements includes a plurality of electric heating elements surrounding at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member. [This invention 1043] The housing assembly of the present invention 1042, wherein at least two of the plurality of electric heating elements are electrically coupled to each other. [This invention 1044] The housing assembly of invention 1042 or invention 1043, wherein at least one of the plurality of electric heating elements is configured to be electrically connected to a power source. [This invention 1045] A housing assembly of any of inventions 1019 to 1044, wherein the housing assembly further includes a jacket member including a heating element or at least one of a plurality of heating elements, the jacket member being configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member. [The present invention 1046] The housing assembly of the present invention 1045, wherein the jacket members are removably connected together to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. [This invention 1047] The housing assembly of claim 1045 or 1046, wherein the jacket member is configured to surround at least a portion of the sidewall member, and optionally to completely surround the sidewall member. [This invention 1048] A housing assembly of any of inventions 1045 to 1047, wherein the jacket member includes two or more jacket parts that are configured together to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the side wall member, optionally completely surround the side wall member. [This invention 1049] The housing assembly of any one of claims 1045 to 1048, wherein a portion of one or more inlets of the inlet housing member and / or a portion of one or more outlets of the outlet housing member are exposed by a jacket member. [The present invention 1050] The housing assembly of any one of claims 1045 to 1049, wherein a portion of one or more inlets of the inlet housing member and / or a portion of one or more outlets of the outlet housing member is outside the jacket member. [This invention 1051] A housing assembly of any of inventions 1045-1050, wherein the heating element or at least one of the plurality of heating elements is a heating flow path including an inlet and an outlet for a heated fluid, and the jacket member includes at least one opening for the inlet for the heated fluid and at least one opening for the outlet for the heated fluid. [This invention 1052] The housing assembly of any of inventions 1045-1050, wherein the heating element or at least one of the plurality of heating elements is an electric heating element, and the jacket member is arranged such that the electric heating element is configured to be electrically connected to a power source. [This invention 1053] The housing assembly of any one of inventions 1048-1052, wherein two or more jacket parts are configured to be detachably connected together. [This invention 1054] A housing assembly according to any one of claims 1048 to 1053, wherein the jacket member includes a plurality of heating elements, and at least two of the two or more jacket parts each include at least one of the plurality of heating elements. [This invention 1055] The housing assembly of the present invention 1054, wherein at least two of the two or more jacket components each further include a temperature sensor. [This invention 1056] one or more jacket components configured to surround at least a portion of the chromatography column; one or more heating elements configured in the one or more jacket components to provide heat to the chromatography column; A jacket member for column chromatography comprising: [This invention 1057] The jacket member of the present invention 1056, wherein one or more jacket components are configured to be removably connected together and surround at least a portion of the chromatography column. [This invention 1058] The jacket member of invention 1056 or invention 1057, wherein the chromatography column is configured to accommodate a stationary phase. [This invention 1059] The jacket member of any of claims 1056 to 1058, wherein one or more heating elements are configured to be part of a temperature control member, the temperature control member being configured to regulate or maintain the temperature of the chromatography column. [The present invention 1060] The jacket member of the present invention 1059, wherein the temperature control member is configured to heat the chromatography column and / or stationary phase to a target temperature of about 30°C to about 39°C, optionally to 37°C or about 37°C. [This invention 1061] The jacket member of claim 1059 or claim 1060, wherein the temperature control member is further configured to maintain the chromatography column and / or stationary phase at a target temperature. [This invention 1062] The jacket member of any one of claims 1056 to 1061, further comprising a temperature sensor configured to measure the temperature of the chromatography column and / or the stationary phase. [This invention 1063] The jacket member of any one of claims 1059 to 1062, wherein the temperature control member includes a heat source. [This invention 1064] The jacket member of any one of claims 1059 to 1062, wherein the temperature control member is configured to be operatively connected to a heat source external to the jacket member. [This invention 1065] The jacket member of any one of claims 1056 to 1064, configured to allow an insulating layer to be disposed between one or more jacket components and at least a portion of the chromatography column. [The present invention 1066] The jacket member of any of claims 1056 to 1065, further comprising an insulating layer configured to be disposed between one or more jacket components and at least a portion of the chromatography column. [This invention 1067] The jacket member of any of claims 1056-1066, wherein one or more heating elements are configured to uniformly heat the chromatography column and / or stationary phase in one or more jacket components. [The present invention 1068] The jacket member of any one of claims 1056 to 1067, wherein each of the one or more jacket components includes at least one of the one or more heating elements. [The present invention 1069] The jacket member of any one of claims 1056 to 1068, wherein the one or more heating elements are each selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. [The present invention 1070] A jacket member according to any one of claims 1056 to 1069, wherein one or more heating elements include a heating flow path including an inlet and an outlet for a heated fluid, and optionally the heated fluid is heated water. [This invention 1071] The jacket member of the present invention 1070, wherein the inlet is configured to connect to an external reservoir of heated fluid. [This invention 1072] The jacket member of any one of claims 1056 to 1071, wherein the one or more heating elements include an electric heating element, and optionally the electric heating element includes a metal plate. [This invention 1073] The jacket member of the present invention 1072, wherein the electric heating element is configured to be electrically connected to a power source. [This invention 1074] The jacket member of any one of claims 1056 to 1073, wherein the jacket member comprises two or more heating elements. [This invention 1075] A jacket member of the present invention 1074, wherein two or more heating elements are configured to be evenly or nearly evenly distributed around the circumference of the chromatography column in one or more jacket components. [This invention 1076] A jacket member according to any one of claims 1056 to 1075, wherein the jacket member comprises two or more heating flow paths each including an inlet and an outlet for a heated fluid, and optionally the heated fluid is heated water. [This invention 1077] The jacket member of the present invention 1076, wherein two or more heating channels are configured to be fluidly connected to one another. [This invention 1078] A jacket member according to any one of claims 1056 to 1077, comprising two or more electric heating elements, optionally two or more electric heating elements comprising metal plates. [This invention 1079] The jacket member of the present invention 1078, wherein two or more electric heating elements are configured to be electrically coupled to each other. [The present invention 1080] The jacket member of claim 1078 or 1079, wherein at least one of the two or more electric heating elements is configured to electrically connect to a power source. [This invention 1081] The jacket member of any one of claims 1056 to 1080, wherein the one or more jacket components include two or more jacket components. [This invention 1082] a chromatography column including an interior cavity configured to accommodate a stationary phase; A jacket member according to any one of claims 1056 to 1081 of the present invention, configured to surround at least a portion of the chromatography column; A housing assembly for column chromatography comprising: [This invention 1083] The housing assembly of the present invention 1082, wherein the chromatography column comprises an inlet housing member, an outlet housing member, and a sidewall member, the inlet housing member, the outlet housing member, and the sidewall member forming an interior cavity. [This invention 1084] The housing assembly of invention 1082 or invention 1083, wherein the jacket members are removably connected together to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. [This invention 1085] The housing assembly of any of claims 1082 to 1084, further comprising a connector configured to functionally and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity. [This invention 1086] A housing assembly set including a plurality of housing assemblies according to any one of the present inventions 1001 to 1085. [This invention 1087] The housing assembly set of the present invention 1086, wherein at least two of the plurality of housing assemblies are arranged in series. [This invention 1088] The housing assembly set of the present invention 1086 or the present invention 1087, wherein at least two of the plurality of housing assemblies are arranged in parallel. [This invention 1089] A chromatography system comprising a housing assembly of any one of the present inventions 1001 to 1055 and the present inventions 1082 to 1085 and at least one additional chromatography column. [The present invention 1090] A chromatography kit comprising a housing assembly of any one of inventions 1001 to 1085, a housing assembly set of any one of inventions 1086 to 1088, or a chromatography system of invention 1089, and a stationary phase for chromatography. [This invention 1091] A chromatography kit comprising the jacket member of any one of 1056 to 1081 of the present invention, a chromatography column, and a stationary phase for chromatography. [This invention 1092] A chromatography column or chromatography column set comprising any one of the housing assemblies of the present invention 1001 to 1085, any one of the housing assembly sets of the present invention 1086 to 1088, or the chromatography system of the present invention 1089, wherein one or more internal cavities of the housing assemblies contain a stationary phase for chromatography. [This invention 1093] A chromatography column comprising the jacket member of any one of 1056 to 1081 of the present invention and a chromatography column, wherein the internal cavity of the chromatography column contains a stationary phase for chromatography. [This invention 1094] A chromatography column set comprising at least one jacket member according to any one of 1056 to 1081 of the present invention and a plurality of chromatography columns, the internal cavity of each of which contains a stationary phase for chromatography. [This invention 1095] The set of chromatography columns of the present invention 1094, wherein a plurality of chromatography columns are arranged in series or parallel, and optionally, a plurality of chromatography columns are operatively connected. [This invention 1096] The chromatography column set of invention 1094 or invention 1095, wherein the plurality of chromatography columns includes a first chromatography column and a second chromatography column, and at least one jacket member is configured to surround the second chromatography column. [This invention 1097] The chromatography kit, chromatography column, or chromatography column set of any one of 1090 to 1096, wherein the stationary phase comprises a gel filtration matrix. [This invention 1098] The chromatography kit, chromatography column, or chromatography column set of any one of 1090 to 1096, wherein the stationary phase comprises an affinity chromatography matrix. [This invention 1099] The chromatography kit, chromatography column, or chromatography column set of any one of 1090 to 1098 of the present invention, wherein the stationary phase comprises a non-magnetic material, a non-ferromagnetic material, or a paramagnetic material, or is a non-magnetic material, a non-ferromagnetic material, or a paramagnetic material. [The present invention 1100] 1090-1099. The chromatography kit, chromatography column, or chromatography column set of any one of claims 1090 to 1099, wherein the stationary phase comprises a selected substance immobilized thereon. [The present invention 1101] 1100. The chromatography kit, chromatography column or chromatography column set of the present invention, wherein the selection agent has the ability to specifically bind to a selection marker on the surface of one or more cells. [The present invention 1102] The chromatography kit, chromatography column or chromatography column set of the present invention 1101, wherein the one or more cells are immune cells. [The present invention 1103] The chromatography kit, chromatography column or chromatography column set of the present invention 1102, wherein the one or more cells are T cells. [The present invention 1104] the selected agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and / or the selection agent comprises an antibody fragment; the selection agent is or comprises a Fab fragment, the selected agent is or comprises a single domain antibody, optionally a VHH antibody, The selected substance is F(ab') 2 a bivalent antibody fragment selected from the group consisting of a bivalent antibody fragment, ... the selected agent is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment and an scFv; and / or the selected agent is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins and avimers; The chromatography kit, chromatography column or chromatography column set according to any one of 1101 to 1103 of the present invention. [This invention 1105] the selection marker is a T cell coreceptor; the selectable marker is or comprises a member of the T cell antigen receptor complex, the selection marker is or comprises the CD3 complex, the selectable marker is or comprises the CD3 chain; the selectable marker is or comprises a CD3γ, CD3δ, CD3ε, or CD3ζ chain; the selection marker is or comprises CD8, the selection marker is or comprises CD4; the selection marker is or comprises CD45RA, the selection marker is or comprises CD27; the selection marker is or comprises CD28, and / or the selection marker is or comprises CCR7; The chromatography kit, chromatography column or chromatography column set according to any one of 1101 to 1104 of the present invention. [The present invention 1106] A chromatography kit, chromatography column, or chromatography column set according to any one of claims 1101 to 1105, wherein the specific binding between the selection substance and the selection marker does not induce a signal to T cells, or does not induce any stimulatory, activation, or proliferation signal to T cells. [This invention 1107] The chromatography kit, chromatography column, or chromatography column set of any one of claims 1101 to 1106, wherein the selected substance comprises anti-CD3 Fab, anti-CD8 Fab, anti-CD4 Fab, or anti-CD27 Fab, or is anti-CD3 Fab, anti-CD8 Fab, anti-CD4 Fab, or anti-CD27 Fab. [This invention 1108] The chromatography kit, chromatography column or chromatography column set of any one of 1101 to 1107, wherein the selection substance is indirectly bound to the stationary phase via a selection reagent to which the selection substance reversibly binds. [This invention 1109] 1108. A chromatography kit, chromatography column or chromatography column set of the present invention, wherein the selection reagent comprises a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide or is a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide. [The present invention 1110] The chromatography column or set of chromatography columns of any of claims 1092 to 1109, further comprising one or more stimulatory substances capable of delivering a stimulatory signal, optionally immobilized on a stationary phase, optionally indirectly immobilized via a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide. [The present invention 1111] The chromatography kit of any one of the present inventions 1090, 1091 and 1097 to 1109, further comprising a stimulating reagent containing one or more stimulating substances capable of delivering a stimulating signal. [The present invention 1112] The chromatography kit, chromatography column or chromatography column set of the present invention 1110 or 1111, wherein the stimulatory signal is from a TCR / CD3 complex in a T cell, a CD3-containing complex in a T cell and / or an ITAM-containing molecule in a T cell. [The present invention 1113] A chromatography kit, chromatography column or chromatography column set of any of claims 1110 to 1112, wherein the one or more stimulating substances include a first stimulating substance capable of delivering a stimulating signal, and the one or more stimulating substances further include one or more second stimulating substances capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating substance. [This invention 1114] The chromatography kit, chromatography column or chromatography column set of the present invention 1113, wherein at least one of the second stimulatory substances has the ability to specifically bind to one or more costimulatory molecules on T cells. [This invention 1115] The chromatography kit, chromatography column or chromatography column set of the present invention 1113 or 1114, wherein the first stimulating substance is anti-CD3 Fab and the second stimulating substance is anti-CD28 Fab. [The present invention 1116] The stimulating reagent comprises a plurality of streptavidin or streptavidin mutein molecules, and the size of the stimulating reagent is (i) greater than 50 nm in radius, (ii) at least 5×10 6 The chromatography kit of any of claims 1111 to 1117, comprising (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per stimulation reagent, and / or (iv) a molecular weight of at least 100 streptavidin tetramers or streptavidin mutein tetramers per stimulation reagent. [This invention 1117] The chromatography kit, chromatography column, or chromatography column set of any one of claims 1113 to 1117, wherein the first stimulating substance and the second stimulating substance independently further comprise a streptavidin-binding peptide. [This invention 1118] Streptavidin-binding peptides TIFF0007746263000005.tif41141 The chromatography kit, chromatography column, or chromatography column set of any one of claims 1109 to 1116, selected from the group consisting of: [This invention 1119] The streptavidin mutein contains the amino acid sequence Val at a sequence position corresponding to positions 44-47 of SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47 or the streptavidin mutein comprises the amino acid sequence Ile at a sequence position corresponding to positions 44-47 of SEQ ID NO:1. 44 -Gly 45 -Ala 46 -Arg 47 The chromatography kit, chromatography column, or chromatography column set of any one of claims 1109 to 1118, comprising: [The present invention 1120] The chromatography kit, chromatography column, or chromatography column set of any one of claims 1109 to 1119, wherein the N-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 10 to 16 of SEQ ID NO:1, and the C-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 133 to 142 of SEQ ID NO:1. [This invention 1121] The chromatography kit, chromatography column or chromatography column set of any one of claims 1109 to 1120, wherein the streptavidin mutein comprises an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, 27, 28, 104 and 105. [This invention 1122] A device comprising any one of the housing assemblies of inventions 1001 to 1055 and inventions 1082 to 1085, any one of the housing assembly sets of inventions 1086 to 1088, or any one of the chromatography systems, chromatography kits, chromatography columns or chromatography column sets of inventions 1089 to 1121, and further comprising an input composition reservoir operably connected to the internal cavity via an inlet of the inlet housing member. [This invention 1123] A device comprising a jacket member according to any one of claims 1056 to 1081 and a chromatography column or set of chromatography columns comprising an internal cavity configured to accommodate a stationary phase for chromatography, and further comprising an input composition reservoir operatively connected to an inlet of the internal cavity to enable uptake of an input composition contained in the input composition reservoir into the internal cavity. [This invention 1124] The device of invention 1122 or invention 1123, further comprising an output composition reservoir operatively connected to an outlet of the internal cavity to enable or effect discharge of the output composition contained in the output composition reservoir from the internal cavity. [Invention 1125] Any of the devices of the present invention 1122 to 1124 in a closed system or a sterile system. [The present invention 1126] A method for on-column stimulation of T cells, comprising the steps of: Incubating a sample containing a plurality of T cells with one or more stimulatory substances to deliver a stimulatory signal to one or more T cells among the plurality of T cells immobilized on a stationary phase in the chromatography column or chromatography column set of any of Inventions 1092 to 1110, Inventions 1112 to 1115, and Inventions 1117 to 1121; After the start of incubation, harvesting the one or more T cells from the stationary phase, thereby producing an output composition comprising stimulated T cells. [This invention 1127] 1126. The method of claim 1126, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and wherein immobilization of said one or more T cells on said stationary phase is achieved by specific binding of said selection agent to said selection marker expressed by said one or more T cells. [This invention 1128] A method for on-column stimulation of T cells, comprising the steps of: (a) adding a sample containing a plurality of T cells to an internal cavity of a chromatography column or a chromatography column set of any of the present inventions 1092 to 1110, the present inventions 1112 to 1115, and the present inventions 1117 to 1121, the internal cavity comprising a stationary phase containing a selection substance that binds to a selection marker on the surface of one or more of the plurality of T cells, thereby immobilizing one or more of the plurality of T cells on the stationary phase; (b) adding to the stationary phase in the chromatography column or set of chromatography columns a stimulation reagent comprising one or more stimulatory substances capable of delivering a stimulation signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulation reagent with the one or more T cells; and (c) harvesting the one or more T cells from the stationary phase after the start of incubation, thereby producing a composition comprising stimulated T cells. [This invention 1129] A method for on-column stimulation of T cells, comprising the steps of: (a)(i) a sample comprising a plurality of T cells; (ii) a stationary phase comprising a selection substance capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells in any one of the chromatography kits of the present invention 1090, the present invention 1091, the present inventions 1097 to 1109, and the present inventions 1111 to 1121; wherein immobilization of the plurality of T cells on the stationary phase is achieved by specific binding of the selection agent to a selection marker; and (b) adding to the stationary phase a stimulating reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; wherein the mixing step and / or the adding step is performed inside or outside the internal cavity of the chromatography column or chromatography column set of the chromatography kit; and (c) harvesting the one or more T cells from the stationary phase after the start of incubation, thereby producing a composition comprising stimulated T cells. [The present invention 1130] 1129. The method of any of claims 1126 to 1129, wherein the one or more T cells are collected from the stationary phase within 24 hours of the start of incubation. [This invention 1131] 11. The method of any of claims 1126-1130, wherein the one or more T cells are harvested from the stationary phase within 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours after the start of incubation. [This invention 1132] 1132. The method of any of claims 1126-1131, wherein the one or more T cells are collected from the stationary phase by gravity flow. [This invention 1133] The method of claim 1132, wherein the gravity flow collection comprises applying a wash medium to a chromatography column or housing assembly containing the stationary phase. [This invention 1134] the wash medium does not contain a competitor or free binder for eluting one or more T cells from the stationary phase; and / or the collecting step is performed without adding a competitor or free binding agent to elute the T cell or cells from the stationary phase; The method of the present invention 1133. [This invention 1135] The method of claim 1134, wherein the competitor or free binder is an agent that competes for binding of the streptavidin-binding peptide of the selection agent to the streptavidin mutein immobilized on the stationary phase. [This invention 1136] 1135. The method of claim 1135, wherein the competitor or free binder is biotin or a biotin analog, and optionally, the biotin analog is D-biotin. [This invention 1137] The stimulatory agent comprises or is an oligomeric stimulatory reagent comprising (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulatory agents capable of delivering a stimulatory signal in one or more T cells, wherein the size of the oligomeric stimulatory reagent is: (i) a radius of greater than 50 nm; (ii) at least 5×10 6 g / mol and / or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulation reagent. [This invention 1138] The streptavidin mutein has the amino acid sequence Val at a sequence position corresponding to positions 44 to 47 in streptavidin in the amino acid sequence shown in SEQ ID NO: 1. 44 -Thr45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 Contains, or The streptavidin mutein has the amino acid sequence Val at a sequence position corresponding to positions 44 to 47 in streptavidin in the amino acid sequence shown in SEQ ID NO: 1. 44 -Thr 45 -Ala 46 -Arg 47 Including, Any of methods 1135 to 1137 of the present invention. [This invention 1139] 1126-1138. The method of any of claims 1126-1138, wherein at least one of the one or more stimulatory agents is capable of delivering a stimulatory signal, the stimulatory signal being from a TCR / CD3 complex in the T cell, a CD3-containing complex in the T cell, and / or an ITAM-containing molecule in the T cell. [The present invention 1140] The method of claim 1139, wherein at least one of the one or more stimulating substances is a first stimulating substance capable of delivering a stimulating signal, and the one or more stimulating substances further comprise one or more second stimulating substances capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating substance. [This invention 1141] The method of claim 1140, wherein the second stimulatory agent is capable of specifically binding to one or more costimulatory molecules on T cells. [This invention 1142] 1141. The method of claim 1141, wherein the costimulatory molecule is selected from among CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. [This invention 1143] The method of any of claims 1140 to 1142, wherein the second stimulatory substance has the ability to specifically bind to CD28 and / or the costimulatory molecule is CD28. [This invention 1144] The method of any of claims 1140 to 1143, wherein the first stimulatory agent specifically binds to CD3 and the second stimulatory agent specifically binds to CD28. [Invention 1145] The method of any of claims 1140 to 1144, wherein the first stimulatory agent comprises a monovalent antibody fragment that binds to CD3, and the second stimulatory agent comprises a monovalent antibody fragment that binds to CD28. [Invention 1146] 1145. The method of claim 1145, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment and a single-chain Fv fragment (scFv). [This invention 1147] The method of any one of claims 1140 to 1146, wherein the first stimulatory agent is anti-CD3 Fab and the second stimulatory agent is anti-CD28 Fab. [This invention 1148] The method of any of claims 1126 to 1147, wherein the temperature control member regulates the temperature of the stationary phase to a target temperature of about 30°C to about 39°C during at least a portion of the incubation. [This invention 1149] The method of any of claims 1126 to 1148, wherein the temperature control member maintains the temperature of the stationary phase at a target temperature of about 30°C to about 39°C during at least a portion of the incubation. [This invention 1150] The method of claim 1148 or claim 1149, wherein the target temperature is about 30°C to about 39°C, optionally 37°C or about 37°C. [This invention 1151] The method of any of claims 1126-1150, wherein the connector allows gas to be introduced into the interior cavity during at least a portion of the incubation. [This invention 1152] 1151. The method of claim 1151, wherein the gas is sterile and is or comprises air. [This invention 1153] The method of claim 1151 or claim 1152, wherein the uptake of gas into the internal cavity is intermittent or continuous during incubation. [This invention 1154] The method of any of claims 1126 to 1153, further comprising the step of introducing a recombinant nucleic acid molecule encoding a recombinant protein into the stimulated T cells of said composition, thereby producing a composition comprising the transduced T cells. [This invention 1155] 1154. The method of claim 1154, wherein the recombinant protein is an antigen receptor. [Invention 1156] 1154. The method of claim 1154, wherein the recombinant protein is a chimeric antigen receptor. [This invention 1157] The method of any one of claims 1154 to 1156, wherein the introduction of the recombinant nucleic acid encoding the recombinant protein is by transduction using viral particles. [This invention 1158] The method of any of claims 1154 to 1157, further comprising the step of incubating the composition comprising the transduced cells under conditions for viral integration. [This invention 1159] The method of any of claims 1154 to 1158, further comprising culturing the composition comprising the transduced cells under conditions for expanding the T cells. [The present invention 1160] The method of any one of claims 1126 to 1159, wherein at least one or all of the steps of the method are performed in a closed system, and optionally, the closed system is automated. [This invention 1161] 1160. The method of any of claims 1126 to 1160, wherein the sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T-cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. [This invention 1162] The method of claim 1161, wherein the apheresis product or leukapheresis product is freshly isolated from the subject. [This invention 1163] The method of claim 1161, wherein the apheresis product or leukapheresis product is thawed from a cryopreserved apheresis product or leukapheresis product. [Brief explanation of the drawings]

[0152] [Figure 1A] 1A and 1B are schematic diagrams of an exemplary housing assembly for column chromatography. Fig. 1A shows an exemplary housing assembly including a temperature control element including a heating coil with an inlet and an outlet for an external hot water supply and a gas supply connector for a threaded air filter. Fig. 1B shows an exemplary housing assembly in an exemplary column chromatography system. [Figure 1B] See legend to Figure 1A. [Figure 2]FIG. 2 is a schematic diagram of an exemplary embodiment of a process for stimulating and selecting target cells. Here, stimulation is performed by incubation of cells, which occurs at least in part in the presence of a support 36 (depicted here as a stationary phase). Components of a selection reagent 31 for cell selection are immobilized on the support (Panel A). The selection reagent 31 has a binding site for a selection agent 32, which is capable of binding to a molecule (selection marker) 34 present on some or all of the target cells. The selection agent 32 is added to the support on which the selection reagent 31 is immobilized under conditions such that the selection agent and the selection agent reversibly bind, e.g., via the binding site, to form an oligomeric complex on which the selection agent multimerizes (Panel B). The selection agent can include two or more agents. Alternatively, a reversibly bound complex of the selection agent and the selection reagent can be added as a complex to the stationary phase for immobilization. As shown, cells 33, including target cells, are mixed with the stationary phase and the multimerized selection agent complex. This causes the target cells to become reversibly immobilized to a support 36 via the selection agent 32 and reagent (selection marker) 34 (Panel C). Optionally, unbound cells are removed prior to or after the addition of a stimulus. A complex containing multimerized stimulator 35 reversibly bound to an oligomeric stimulator reagent 37 is added under conditions such that the stimulus 35 specifically binds to a molecule on the target cells, thereby inducing or modulating a signal in the immobilized target cells expressing the marker (Panel D). [Figure 3]Figures 3A and 3B show the results of WST metabolic assays of T cells from three different donors incubated with anti-CD3 / anti-CD28 multimerized on oligomeric reagents of different batches. Figure 3A summarizes the WST metabolic activity (as indicated by the WST ratio) for all tested batches (pools) compared with a reference batch containing anti-CD3 / anti-CD28 multimerized on an oligomeric backbone with a mean hydrodynamic radius of 36 nm or 101 nm. The average WST metabolic activity (as indicated by the WST ratio) in T cells from different donors for each tested batch and reference reagent is shown in Figure 3B. [Figure 4] FIG. 4 is a schematic representation of an exemplary on-column T cell selection and stimulation process. [Figure 5] Figure 5 shows that the elution efficiency using the exemplary heat / gas column with heating and gas delivery elements was approximately twice that of the reference column. The estimate (gray bar) is the theoretical number of captured cells that could be eluted assuming 100% efficiency. [Figure 6] Figure 6 depicts flow cytometry quantification of cells in the starting material, negative fraction, or positive fraction after on-column T cell selection and stimulation using an exemplary column with heating and gas delivery elements. Cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, CD45, and CD14. [Figure 7A] Figures 7A and 7B show the results of T cells after on-column selection and stimulation using an exemplary column with heating and gas delivery elements. During this subsequent incubation, cells were monitored by flow cytometry on days 1, 2, and 3 for cell number and cell surface expression after staining the cells with antibodies recognizing CD3, CD4, CD8, and the activation markers CD69 and CD25. The flow cytometry results are shown in Figure 7A. Assessment of cell number and fold expansion after subsequent incubation revealed that the selected and stimulated T cells began to increase in number on day 3, consistent with the cells' proliferative potential, as shown in Figure 7B. [Figure 7B] See legend to Figure 7A. [Figure 8] Figures 8A-8C show the results of on-column T cell selection using an exemplary heat / gas column, using a cryopreserved apheresis sample as the starting sample. Figure 8A shows that cryopreserved apheresis samples (CAPH) generally have a higher monocyte content (>20% as a percentage of live CD45+ cells) compared to fresh apheresis samples (APH). Figure 8B shows the percentage of CD3- or CD14-positive cells in the starting material and the positive fraction. The number of T cells selected using the chromatography column is shown in Figure 8C. Here, two sequential rounds of selection for CD3 were performed. [Figure 9] FIG. 9 is a schematic diagram of a selection and stimulation run using two exemplary identical heat / gas columns arranged in series (Run 1) and two exemplary identical heat / gas columns arranged in parallel (Run 2). [Figure 10A] Figures 10A and 10B compare the results of T cell selection and stimulation in Run 1 and Run 2. Figure 10A shows flow cytometry analysis of the starting material, negative fraction, and positive fraction, in which cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fraction were harvested and incubated. The left panel of Figure 10B shows the expression of activation markers CD25 and CD69 in cells on day 1 of incubation. Representative results of cell number during incubation in Run 1 (■) and Run 2 (●) are shown in the right panel of Figure 10B. [Figure 10B] See legend to Figure 10A. [Figure 11A]Figures 11A and 11B show the results of on-column T cell selection by CD3 selection and stimulation in two exemplary heat / gas columns arranged in parallel, using a concentrated blood sample as the starting material. Figure 11A shows flow cytometry analysis of the starting material, negative fraction, and positive fraction, in which cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fraction were harvested and incubated. CD4 / CD8 and CD25 / CD69 expression of the incubated cells is shown in Figure 11B. [Figure 11B] See legend to Figure 11A. [Figure 12] Figure 12 shows the results of an exemplary process for selecting T cells directly from whole blood using Sephadex® G-50 as the resin in an exemplary thermal / gas chromatography column. The starting material, negative fraction, and positive fraction from this CD3+ T cell selection were stained with propidium iodide (PI) and CD3 antibody and quantified by flow cytometry. [Figure 13] Figure 13 shows the effect of 24 hours of on-column stimulation with anti-CD3 / anti-CD28 oligomer stimulating reagent on the surface expression (assessed as mean fluorescence intensity, MFI) of CD3, CD4, and CD8 molecules when these molecules were used as selection markers to immobilize cells on the stationary phase of a chromatography column. The surface expression pattern is compared to a control condition without on-column stimulation with anti-CD3 / anti-CD28 oligomer stimulating reagent. Cells were isolated from apheresis samples applied to the stationary phase. [Figure 14] Figure 14 shows exemplary kinetics of downregulation and re-expression of TCR / CD3 complexes upon on-column stimulation with anti-CD3 / anti-CD28 oligomer stimulation reagents, when CD3 was used as a selection marker to immobilize cells on the column. Cells were isolated from apheresis samples applied to the stationary phase. CD3 / TCR complexes were assessed using antibodies against the alpha-beta TCR chain. [Figure 15A]Figures 15A-15B show the phenotypic and functional characteristics of cultured T cells spontaneously shed during on-column stimulation with anti-CD3 / anti-CD28 oligomer stimulating reagent. Figure 15A, from left to right, shows T cell size and CD3, CD69, and CD25 expression 24 hours and 5 days after on-column stimulation. Figure 15B shows the proliferative potential of spontaneously shed cultured T cells, as indicated by cell count and fold expansion. Cells were isolated from apheresis samples applied to the stationary phase and collected using a washing step. [Figure 15B] See legend to Figure 15A. [Figure 16A] Figures 16A-16D show exemplary effects of incubating T cells with anti-CD3 / anti-CD28 oligomer stimulatory reagents in the presence or absence of Compound 63 on mTor signaling and viability and growth kinetics. Figure 16A shows pS6 expression by memory subsets in live CD8+ T cells. Figure 16B shows the mean fluorescence intensity (mfi) of pS6 expression in total CD8 T cells with the indicated treatments. Figures 16C-16D show viability and total T cell numbers, respectively, in culture over time (shown in days, e.g., d1) after the start of stimulation ("input"). In Figures 16C-16D, the black lines correspond to T cell compositions incubated in the presence of Compound 63, and the gray lines correspond to T cell compositions incubated in the absence of Compound 63. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17-1]Figures 17A-17F show exemplary functional and phenotypic characteristics of cryopreserved CAR-T cells generated using a method employing incubation with anti-CD3 / anti-CD28 oligomer stimulating reagents in the presence or absence of compound 63. Figure 17A shows intracellular expression of caspases upon thawing. Figures 17B and 17D show phenotypic profiles of CD8 and CD4 CAR-T cells, respectively, by subset expression of CD27 and / or CCR7. Figures 17C and 17E show intracellular IL2, IFNg, or TNF (left panels) or a combination of IL2 and / or IFNg or TNF (right panels) in CD8 and CD4 CAR-T cells, respectively, stimulated with antigen-bearing targets. Figure 17F shows expansion and survival over 12 days (left panel) and the total expansion metric calculated by the area under the growth curve (AUC, right panel) for CAR-T cells stimulated with anti-CAR beads. [Figure 17-2] See description of Figure 17-1. [Figure 18A] FIG. 18A shows CD3+, CD4+, and CD8+ T cell yields after cell selection using the on-column stimulation process described in Example 11 or an alternative process. [Figure 18B] 18B-18C show the total number of cells (FIG. 18B) and percentage of viable cells (FIG. 18C) recovered after using the on-column stimulation process described in Example 11 or an alternative process. [Figure 18C] See legend to Figure 18B. [Figure 19A] Figures 19A-19D represent the percentage of viable cells (e.g., purity; Figure 19A), the percentage of viable cells expressing an exemplary CAR (Figure 19B), the percentage of viable cells expressing CD4 at the time of selection and at day 8 of the process (Figure 19C), and the distribution (percentage) of T cell phenotypes for each donor at day 5 of culture (day 8 from the start of the process) for the on-column stimulation process described in Example 11 or an alternative process (Figure 19D). [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 20] Figure 20 depicts CD19+ HEK cell lysis over time during culture with anti-CD19 CAR T cells modified using the on-column stimulation process described in Example 11 or an alternative process, and under control conditions. [Figure 21A] Figures 21A-21C show antigen-specific CAR T cell IFNg (Figure 21A), IL-2 (Figure 21B), and TNFα (Figure 21C) production for CD4 and CD8 T cells engineered using the on-column stimulation process described in Example 11 or an alternative process. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 22A] Figures 22A-22C show the CD4:CD8 ratio (Figure 22A), transduction efficiency (CD4 and CD8 cells combined; Figure 22B), and percentage of viable cells (Figure 22C) of modified T cells generated using the on-column stimulation process or an alternative process described in Example 11. Three production runs are shown for each process. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 23] Figure 23 depicts tumor size by mean radiance across all treatment groups 6 days after mice were injected (iv) with a B cell lymphoma cell line (Raji) and before they were treated with CAR-T cell compositions. Treatment groups refer to CAR-T cell compositions produced by each of three manufacturing runs of the on-column stimulation process or an alternative process described in Example 11. [Figure 24]Figure 24 depicts tumor burden in mice injected with a B cell lymphoma cell line (Raji) over time for each treatment group. The effect of CAR T cell treatment is shown for the on-column stimulation process described in Example 11 or the alternative process, and for each of the three manufacturing runs (see Figures 22A-22C). [Figure 25] Figures 25-28 are schematic diagrams of an exemplary column chromatography housing assembly. The exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member surrounding the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of two jacket parts, each of which includes a heating coil with an inlet and an outlet for an external hot water supply. The two jacket parts collectively form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 25 is an exploded view of an exemplary housing assembly. Figures 26A-26C are views of the interior (Figure 26A), side (Figure 26B), and exterior (Figure 26C) of one jacket part. Figure 27 is a view of an exemplary housing assembly, showing the inlet for the external hot water supply and a portion of the inlet of the inlet housing member. 28 is a diagram of an exemplary housing assembly showing an outlet for an external hot water supply and a portion of the outlet housing member. Optional features (not shown) of this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase from the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase from the outlet of the internal cavity, and a tubing set connector. [Figure 26] See legend to Figure 25. [Figure 27] See legend to Figure 25. [Figure 28] See legend to Figure 25. [Figure 29]Figures 29-31 are schematic diagrams of an exemplary column chromatography housing assembly. The exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member that surrounds the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of three jacket parts, each of which contains an electric heating element including a metal plate. The three jacket parts collectively form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 29 is an exploded view of the exemplary housing assembly. Figures 30A-30C are three views of one jacket part. Figure 30D shows the electric heating element. Figure 31 is a view of the exemplary housing assembly, showing the electrical connection points of the electric heating element and a portion of the outlet housing member. Optional features (not shown) of this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase from the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase from the outlet of the internal cavity, and a tubing set connector. [Figure 30] See legend to Figure 29. [Figure 31] See legend to Figure 29. [Figure 32] Figure 32 shows the CD27 surface expression of cells immobilized on the stationary phase of a heated column using CD27 as a selection marker and stimulated on-column with anti-CD3 / anti-CD28 oligomer reagent. The column was heated using a jacket containing two heating coils, each with an inlet and outlet for an external heated water supply. The heated column also included a gas supply connector for a threaded air filter. As a control, CD27-selected cells were not subjected to on-column stimulation with anti-CD3 / anti-CD28 oligomer stimulation reagent. Cells were isolated from an apheresis sample applied to the stationary phase. [Figure 33] Figure 33 shows the surface expression of CD3 and CD27 on cells sequentially isolated from an apheresis sample using two separation columns. CD27 was used as the selection marker in the first column, and the positive fraction from the first column was passed through the second column with the CD3 selection marker. The immobilized cells in the second column were stimulated with an anti-CD3 / anti-CD28 oligomer stimulating reagent. The second column was heated using a jacket member containing two heating coils, each with an inlet and outlet for an external heated water supply. The heated column also contained a gas supply connector for a threaded air filter. [Figure 34A] Figures 34A-34E show CD3+ depletion (Figure 34A), CD4 and CD8 expression (Figure 34B), CD69 expression (Figure 34C), viability (Figure 34D), and viable cell count (Figure 34E) of cells after on-column stimulation in chromatography columns heated using different heating elements. The columns were heated using a jacket containing two heating coils (water) or three metal plates (metal) as electrical heating elements. The columns also included a gas supply connector for a threaded air filter. [Figure 34B] See legend to Figure 34A. [Figure 34C] See legend to Figure 34A. [Figure 34D] See legend to Figure 34A. [Figure 34E] See legend to Figure 34A. DETAILED DESCRIPTION OF THE INVENTION

[0153] Detailed Description In some aspects, a housing assembly for column chromatography is provided herein. In some aspects, the housing assembly for column chromatography includes at least an inlet housing member and an outlet housing member, where the inlet housing member and the outlet housing member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving gas uptake into the internal cavity. In some aspects, a housing assembly for column chromatography is provided herein, including a chromatography column including an internal cavity configured to accommodate a stationary phase; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving gas uptake into the internal cavity. In some embodiments, the chromatography column includes an inlet housing member, an outlet housing member, and a sidewall member, where the inlet housing member, the outlet housing member, and the sidewall member form the internal cavity. In some aspects, the temperature control member includes one or more heating elements. In some aspects, the housing assembly further includes a jacket member including a temperature control member including at least one of the one or more heating elements. In some aspects, the jacket member surrounds at least a portion of the inlet housing member and / or at least a portion of the outlet housing member. In some aspects, provided herein is a jacket member for column chromatography. In some aspects, the jacket member is configured to surround at least a portion of a chromatography column. In some aspects, provided herein is a device including a chromatography column housing assembly and a stationary phase housed therein to form a chromatography column. In some aspects, the stationary phase is configured to immobilize target cells thereon.In some aspects, the device is configured to select and / or stimulate a cell or cell population. In some embodiments, the selected and / or stimulated cell or cells are useful in a cell manufacturing process for genetically modifying one or more cells, for example, for the production of a cellular therapeutic agent.

[0154] Methods for generating cell populations suitable for use in cell therapy, such as selected (enriched) and stimulated cell populations, often require separate selection and stimulation steps, which can lengthen the manufacturing process. A variety of methods are available for generating cell populations suitable for use in cell therapy, such as cells engineered to express a recombinant protein (e.g., a chimeric antigen receptor). However, in some aspects, the use of these reagents or systems can require long or relatively long times to generate cells, at least in part because multiple processing steps must be performed. Multiple processing steps can also result in cell stress, thereby affecting the usefulness of the cells for downstream processing. Furthermore, selection techniques may involve contaminating selected cells with selection-related particles, such as selection agents such as Fab fragments, as well as competing reagents and / or free binding agents used to facilitate cell detachment from the stationary phase, requiring additional wash steps and / or medium changes to purify the output composition. The additional processing steps can result in cell stress, which, in addition to taking a significant amount of time to complete, can potentially affect downstream cell processing or even the biology of the cells. There is a need for additional devices and methods for producing cell compositions.

[0155] In some aspects, provided herein are devices for selecting cells and / or stimulating selected cells from a sample containing target cells (e.g., T cells, such as CD3+, CD4+, or CD8+ T cells), and methods of using the devices. In some aspects, the devices include at least an inlet housing member and an outlet housing member, where the inlet housing member and the outlet housing member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving gas uptake into the internal cavity. In some aspects, the stationary phase is configured to immobilize target cells thereon. In some aspects, the temperature control member includes one or more heating elements. In some aspects, the device further includes a jacket member including a temperature control member including one or more heating elements. In some aspects, the jacket member is configured to surround at least a portion of the inlet housing member and / or at least a portion of the outlet housing member. The devices provided herein also include a jacket member for use in selecting cells from a sample containing target cells (e.g., T cells, such as CD3+, CD4+, or CD8+ T cells) and / or stimulating selected cells. In some aspects, the jacket member includes a temperature control member configured to provide heat to the chromatography column. In some aspects, the jacket member is configured to surround at least a portion of the chromatography column. In some aspects, the jacket member includes one or more heating elements.

[0156] In one aspect, it is found herein that methods for on-column selection and / or stimulation of target cells are improved if the temperature of cells immobilized on a stationary phase in the internal cavity of column chromatography is controlled to maintain a temperature of 37°C or about 37°C, or 37°C ± about 5°C. It is also found herein that column configurations that allow for gas exchange (e.g., the presence of air in the column) improve the overall health, fitness, or condition of cells during on-column selection and / or stimulation. In certain embodiments, provided devices are capable of controlling the temperature within the column (e.g., 37°C or 37°C ± about 5°C) during cell selection and stimulation by provided on-column methods. In certain embodiments, provided devices are capable of controlling the temperature within the column (e.g., 37°C or 37°C ± about 5°C) and allowing gas exchange, e.g., the presence of air, during cell selection and stimulation by provided on-column methods. In some aspects, the provided devices can be used in conjunction with cell selection and / or stimulation methods to facilitate or improve cell activation and detachment or elution of cells from a stationary phase for downstream processing of the cells, such as subsequent genetic modification of the cells.

[0157] In one aspect, the temperature control member is configured to provide a temperature appropriate for selection and / or stimulation of target cells immobilized on the stationary phase in the internal cavity of the column chromatography. To this end, the device or a system including the device can be provided with heating and / or cooling means. In some embodiments, the temperature control member is configured to provide heat to the stationary phase, thereby regulating or maintaining the temperature of the target cells immobilized thereon. In some embodiments, the temperature of the target cells is regulated and / or maintained at an optimal temperature for cell selection and / or stimulation. In one aspect, the temperature control member is configured to maintain the temperature of the target cells immobilized on the stationary phase at an optimal temperature for stimulation with a stimulation reagent. In some embodiments, the optimal temperature for stimulation is greater than about 2°C, greater than about 4°C, greater than about 8°C, greater than about 12°C, greater than about 16°C, greater than about 20°C, greater than about 24°C, greater than about 28°C, greater than about 32°C, or greater than about 36°C. In some embodiments, the optimal temperature for stimulation is 37°C or about 37°C. In some embodiments, the temperature of the target cells immobilized on the stationary phase is maintained at a constant temperature value (e.g., an optimum temperature) during at least a portion of the stimulation. In some embodiments, the temperature of the target cells immobilized on the stationary phase is maintained at a selected temperature value (e.g., an optimum temperature) ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C during at least a portion of the stimulation. In some embodiments, the temperature of the target cells immobilized on the stationary phase is maintained at 37°C ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C during at least a portion of the stimulation.

[0158] In one aspect, the device includes a connector configured to operably connect the internal cavity to a gas source, thereby enabling or achieving uptake of gas into the internal cavity. In one aspect, the gas is present in the internal cavity during at least a portion of stimulation of target cells immobilized on the stationary phase of the chromatography column. In some aspects, the gas includes air.

[0159] In some aspects, the devices and methods provided herein reduce and / or minimize cell handling and processing times during the manufacturing process. In some aspects, the device includes a stimulating agent configured to stimulate immobilized cells on the stationary phase (also referred to herein as on-column stimulation). In some aspects, the device further includes one or more elements, such as a heating element and / or a gas supply element, that promote or facilitate cell activation, thereby promoting or facilitating spontaneous detachment of the selected and stimulated cells from the stationary phase. In some aspects, the device further includes one or more elements configured to collect the selected and stimulated cells that spontaneously detach from the stationary phase (e.g., due to stimulation) without using a competitor or free binder to promote detachment. In some aspects, the devices and methods provided herein can combine the cell selection, stimulation, and / or collection steps. In some aspects, the devices and methods provided herein do not require a separate step to promote detachment of the selected and stimulated cells from the stationary phase. In some aspects, the devices and methods provided herein do not require a separate purification step, such as a step to remove agents used to promote detachment (e.g., competitors and / or free binders). Thus, the devices and methods provided herein reduce the number of processing steps required to produce a composition of selected and stimulated cells suitable for downstream processing (e.g., genetic modification, expansion, subsequent incubation, stimulation, and / or selection (e.g., polishing)), thereby reducing production time, minimizing potential cell stress, and / or reducing the possibility of contamination. In certain embodiments, the devices and methods herein produce an output composition of selected and stimulated cells suitable for downstream processing within a certain period of time, for example, within 24 hours.

[0160] The provided devices and methods can select cells, such as CD3+, CD4+, and CD8+ T cells, from other components, e.g., from other cells in a sample, immobilize the cells on a stationary phase of a chromatography column, stimulate the selected cells immobilized on the stationary phase, and collect the selected and stimulated cells without processing steps to detach the cells from the stationary phase and to remove the agent used to promote detachment from the output composition of the selected and stimulated cells. In certain aspects, the provided devices and methods can produce a population of selected and stimulated cells in a reduced time compared to methods that include separate selection and stimulation steps and require additional steps to detach the cells from the stationary phase and to remove the agent used to promote detachment. In certain aspects, the provided devices and methods can produce an output population (also referred to as a composition) of selected and stimulated cells suitable for downstream processing (e.g., genetic modification, expansion, and / or subsequent incubation, stimulation, and / or selection rounds) within 24 hours of initiating on-column stimulation, also referred to herein as on-column stimulation. In some embodiments, the methods of the devices herein involve the use of a stimulatory agent capable of delivering a stimulatory signal to cells by binding to a molecule on the surface of the cells. In some embodiments, the stimulatory agent is included in an oligomeric stimulatory reagent that can be added to the stationary phase. In some embodiments, this stimulation results in the spontaneous detachment of the selected cells from the stationary phase, thereby allowing for the collection of the selected and stimulated cells without additional processing steps to detach the cells from the stationary phase and to remove agents used to promote detachment from the stimulated cell output composition.In certain aspects, the method successfully produces, within 24 hours of initiating on-column stimulation, a composition of selected, stimulated cells that is uncontaminated (e.g., free of agents used for desorption (e.g., competitors, free binders) and / or selection agents), suitable for further processing, e.g., genetic modification, expansion, incubation, or subsequent rounds of stimulation and / or selection (e.g., polishing).

[0161] All publications mentioned in this application, including patent documents, scientific articles, and databases, are incorporated herein in their entirety for all purposes to the same extent as if each individual publication was individually incorporated herein by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0162] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0163] I. Devices and Kits for Cell Selection, Stimulation and / or Modification In certain aspects, the devices and methods provided herein allow target cells (e.g., CD3+, CD4+, or CD8+ T cells) to be selected and stimulated on the stationary phase of a chromatography column, which stimulation promotes downregulation of the molecule (i.e., selection marker) used for cell selection, resulting in spontaneous cell detachment from the stationary phase. In some embodiments, the stationary phase of the chromatography column is functionalized with an agent (e.g., a selection agent) capable of specifically binding to a molecule (e.g., a selection marker) on the surface of the target cell. Thus, when a sample containing target cells bearing a selection marker (e.g., CD3, CD4, CD8) is mixed with the stationary phase, the target cells (e.g., CD3+, CD4+, CD8+ T cells) are indirectly immobilized to the stationary phase. Exemplary selection agents and selection reagents are described in Sections II-B-1 and II-B-2. In certain aspects, target cells (e.g., T cells) are stimulated while immobilized on the stationary phase (e.g., on-column stimulation), e.g., by the addition of a stimulatory agent, a stimulating reagent comprising the stimulatory agent, and / or via a stimulatory agent directly or indirectly coupled to the stationary phase. Exemplary stimulatory agents and stimulating reagents comprising the stimulatory agent (e.g., oligomeric stimulating reagents) are described in Sections II-B-1 and II-B-2. Thus, in some aspects, the provided methods and other embodiments are advantageous in that they simplify multiple processing steps (e.g., selection and stimulation) and allow the simplified process to occur in the same container and / or the same closed system, which can result in improved efficiency and sterility.

[0164] In certain aspects, the devices and methods provided herein involve the use of oligomeric stimulatory reagents containing stimulatory substances capable of delivering stimulatory signals to target cells (e.g., T cells). Existing reagents used to stimulate T cells in vitro, for example, in the absence of exogenous growth factors or in the presence of small amounts of exogenous growth factors, are known (see, e.g., U.S. Pat. No. 6,352,694 (B1) and European Patent EP 0 700 430 B1). Generally, such reagents may use beads with a diameter of more than 1 μm, e.g., magnetic beads, to which various binding agents (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies) are immobilized. However, such magnetic beads may be difficult to incorporate into methods for stimulating cells, for example, under conditions required for clinical trials or therapeutic purposes. This is because it is necessary to ensure that these magnetic beads are completely removed before administering expanded T cells to a subject. In some aspects, such removal, for example, by exposing the cells to a magnetic field, may reduce the yield of viable cells available for cell therapy. In certain instances, the time that such reagents, e.g., stimulatory reagents containing magnetic beads, are incubated with cells must be minimized to allow a sufficient number of T cells to detach from the reagent. Furthermore, reagents such as beads are not readily compatible with column chromatography due to their physical makeup.

[0165] The devices and methods provided herein that utilize oligomeric stimulation reagents overcome such potential limitations. For example, in some embodiments, the provided methods include adding a soluble oligomeric reagent that is not bound to a solid support (e.g., beads) to the stationary phase to initiate stimulation. In some embodiments, the risk of residual reagent in the output cells generated or produced by the method is reduced or avoided by the use of oligomeric reagents. This is because the addition of a competing reagent or free binder can be used to dissociate (e.g., disrupt) the binding of the stimulator-containing oligomeric stimulation reagent from the cells. In some embodiments, this also means that a process that adheres to GMP standards can be more easily established compared to other methods, such as methods that require additional measures to ensure that the final population for administration is bead-free. Thus, in some aspects, removal or separation of the oligomeric stimulation reagent from the cells, such as by adding a competing reagent or free binder, results in little or no cell loss compared to removal or separation using bead-based stimulation reagents. In some aspects, the timing of removal or separation of the stimulating reagent or oligomeric stimulating reagent is unlimited or less restrictive than the removal or separation of bead-based stimulating reagents. Thus, in some aspects, the stimulating reagent or oligomeric stimulating reagent may be removed or separated from the cells at any time or step during the provided methods.

[0166] In some aspects, the provided devices are improved devices for methods involving the isolation, processing, or manipulation of target cells immobilized on a stationary phase, e.g., methods for on-column selection and / or stimulation of target cells. In some aspects, the provided devices allow for temperature regulation, e.g., heating of cells immobilized on a stationary phase. In some aspects, the provided devices allow for the temperature of immobilized cells to be maintained, e.g., at or about 37°C, or 37°C ± about 5°C. In some aspects, regulating and maintaining the temperature of cells by the provided devices improves on-column stimulation of immobilized cells, e.g., by improving the overall health, fitness, or condition of the immobilized cells during on-column stimulation. In some aspects, the provided devices also allow for gas exchange, e.g., the presence of air in the stationary phase. In some aspects, the gas exchange enabled by the provided devices improves the overall health, fitness, or condition of the immobilized cells during on-column stimulation. Thus, in some aspects, the provided on-column selection and / or stimulation methods used in conjunction with the provided devices provide improved, e.g., healthier, cells for subsequent modification for use in therapy, e.g., autologous cell therapy.

[0167] In some embodiments, the devices provided herein can be used to perform any of the methods described in Section II.

[0168] In certain aspects, the duration of the provided methods can be measured from the time when cells, e.g., T cells of an input cell population or input cell sample, are first contacted with or exposed to stimulatory conditions (e.g., those described in Section II-D herein) (also referred to herein as the start of incubation with a stimulator or under stimulatory conditions), e.g., when exposure to a stimulatory reagent is initiated. In some embodiments, the duration required to collect an output population (also referred to herein as an output composition) containing stimulated target cells (e.g., CD3+, CD4+, CD8+ T cells) is measured from the start of incubation (e.g., addition of or exposure to a stimulatory reagent). In particular embodiments, the duration of incubation is 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours, or about 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours , 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours, or less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours. In some embodiments, the duration of the provided incubation is at or about 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10%, or less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of the alternative or existing process.

[0169] It is contemplated herein that the output composition of selected and stimulated cells may be further processed, for example, the output cells may be genetically modified to express a recombinant protein such as a chimeric antigen receptor, and / or the output cells may be subjected to further incubation, stimulation, expansion, selection (e.g., polishing), and / or formulation.

[0170] In certain embodiments, the methods using the provided devices are performed on a sample, such as, for example, an apheresis sample, a buffy coat sample, or whole blood. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is collected from a human subject. In some embodiments, the biological sample is collected from a patient suffering from a disease or condition. In some embodiments, the method is performed on a population of cells previously isolated, enriched, or selected from the sample, such as a population of CD4+ T cells and CD8+ T cells. In some embodiments, the sample or cells isolated from the sample may be cryopreserved.

[0171] In some embodiments, provided herein are devices, kits, systems, and / or articles of manufacture for selecting, stimulating, and / or modifying cells. In some embodiments, a stationary phase arrangement for chromatography is provided. In some embodiments, the arrangement further includes a bioreactor. The bioreactor is suitable for cell expansion, and the stationary phase is suitable for cell separation and on-column stimulation. In various embodiments, the stationary phase is a gel filtration matrix and / or affinity chromatography matrix, and the gel filtration chromatography matrix and / or affinity chromatography matrix includes a selection reagent, the selection reagent including a binding site Z1 that specifically binds to a binding partner C1 contained in a selection agent, and / or the selection reagent including a binding site Z2 that specifically binds to a binding partner C2 contained in a second selection agent. This makes the stationary phase suitable for immobilizing a first selection agent and / or a second agent, a first binding partner C1, and / or a second binding partner C2 thereon. Additionally, the bioreactor and the stationary phase are fluidically connected. This arrangement can be used in continuous expansion culture and can be incorporated into known cell expansion systems such as the Quantum® Cell Expansion System or the Xuri Cell Expansion System W25.

[0172] In some embodiments, the stationary phase is contained within a chromatography column. The arrangement may further include a second stationary phase fluidly connected to the first stationary phase. The second stationary phase may be a gel filtration and / or affinity chromatography matrix, which may include a selection reagent and be suitable for immobilizing the multimerization reagent on the stationary phase. This type of arrangement can facilitate sequential selection of target cells (e.g., T cells, CD4, CD3, CD8 T cells), one of which may also be suitable for on-column stimulation as described herein.

[0173] In some aspects, provided embodiments are directed to a device for the purification (e.g., selection) and cultivation, e.g., stimulation or expansion, of a composition of cells, the device comprising an arrangement of at least one of a bioreactor as defined above and a first or second stationary phase for chromatography.

[0174] The device may further comprise multiple arrangements of bioreactors and stationary phases fluidly connected in series.

[0175] The device may include a sample inlet fluidly connected to the chromatographic stationary phase. The device may also include a sample outlet for purified stimulated target cells fluidly connected to the last stationary phase of at least one arrangement of bioreactors and chromatographic stationary phases.

[0176] In some embodiments, the device can be designed as a functionally closed system.

[0177] A. Chromatography Housing Assembly In some embodiments, provided herein are chromatography housing assemblies (also referred to herein as column chromatography housing assemblies or housing assemblies) suitable for the chromatography-based cell selection and / or stimulation methods described herein. The chromatography housing assemblies can be provided with or without a chromatographic stationary phase.

[0178] In one aspect, provided herein is a housing assembly for column chromatography, comprising at least an inlet housing member and an outlet housing member, wherein the inlet housing member and the outlet housing member define an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operatively connect the internal cavity to a gas source, thereby enabling or achieving the introduction of gas into the internal cavity. In one aspect, the housing assembly for column chromatography further includes a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form the internal cavity. The connector can be disposed on the inlet housing member, the outlet housing member, and / or the sidewall member. The connector can be a bonded connector, a threaded connector, a Luer connector (e.g., a Luer lock connector or a Luer slip connector), a barbed connector, or any combination thereof. In any of the above embodiments, the connector can be configured to sealingly engage tubing in fluid communication with the gas source. In any of the above embodiments, the connector can include one or more filters and / or the connector can be operatively connected to a tubing system that includes one or more filters. The one or more filters can be gas filters, e.g., air filters. The one or more filters can be sterile filters and / or sterilizing filters for sterile filtration. In one aspect, a gas is present in the internal cavity during at least a portion of the stimulation of target cells immobilized on the stationary phase of the chromatography column. In some aspects, the gas includes air. In one aspect, stimulation of cells (e.g., lymphocytes such as T cells) in the presence of gas (e.g., air) facilitates cell activation and downstream processing of the cells, e.g., detachment or elution of the cells from the stationary phase and / or genetic modification of the cells.

[0179] In some embodiments, the interior cavity of the housing assembly contains a volume of 1 to 40 mL or about 1 to 40 mL, e.g., 1 to 35 mL, 1 to 30 mL, 1 to 25 mL, 1 to 20 mL, 1 to 15 mL, 1 to 10 mL, 1 to 5 mL, 5 to 40 mL, 5 to 35 mL, 5 to 30 mL, 5 to 25 mL, 5 to 20 mL, 5 to 15 mL, 5 to 10 mL, 10 to 40 mL, 10 to 35 mL, 10 to 30 mL, 10 to 25 mL, 10 to 20 mL, 10 to 15 mL, 15 to 40 mL, 15 to 35 mL, 15 to 30 mL, 15 to 25 mL, 15 to 20 mL, 20 to 40 mL, 20 to 35 mL, 20 to 30 mL, 20 to 25 mL, 25 to 40 mL, 25 to 35 mL, 25 to 30 mL, 30 to 40 mL, 30 to 35 mL, mL or 35-40mL, or approximately 1-35mL, 1-30mL, 1-25mL, 1-20mL, 1-15mL, 1-10mL, 1-5mL, 5-40mL, 5-35mL, 5-30mL, 5-25mL, 5-20mL, 5-15mL, 5-10mL, 10-40mL, 10-35mL, 10-30mL, 10-25mL, 10-2 The internal cavity of the housing assembly can accommodate a bed volume of 15-25 mL or about 15-25 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume of 15-20 mL or about 15-20 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume of 15-20 mL or about 15-20 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume of 18-20 mL or about 18-20 mL.

[0180] In some embodiments, a housing assembly for column chromatography includes one or more connectors, for example, two or more connectors. In some embodiments, a housing assembly for column chromatography includes two connectors. In some embodiments, the two or more connectors are disposed on at least the inlet housing member. In some embodiments, the two or more connectors are disposed on at least the outlet housing member. In some embodiments, a connector is disposed on at least each of the inlet and outlet housing members. In some embodiments, both the inlet and outlet housing members have connectors disposed thereon.

[0181] In any of the above embodiments, the temperature control member can be configured to adjust or maintain the temperature of the stationary phase in the internal cavity. In any of the above embodiments, the temperature control member can be configured to heat the stationary phase in the internal cavity from a starting temperature (e.g., room temperature) to a target temperature of about 35°C to about 39°C (e.g., 37°C or about 37°C). In some embodiments, the temperature control member can be configured to heat the stationary phase to a target temperature of about 30°C to about 39°C. In some embodiments, the starting temperature is about 2°C, about 4°C, about 8°C, about 12°C, about 16°C, about 20°C, about 24°C, about 28°C, about 32°C, about 36°C, or greater than about 36°C. In some embodiments, the temperature for stimulation is 37°C or about 37°C. In some embodiments, the target temperature (e.g., the optimal temperature for cell stimulation) is greater than about 2°C, about 4°C or higher, about 8°C or higher, about 12°C or higher, about 16°C or higher, about 20°C or higher, about 24°C or higher, about 28°C or higher, about 32°C or higher, about 36°C or higher, about 37°C or higher, about 38°C or higher, about 39°C or higher, or about 40°C or higher. In some embodiments, the temperature of target cells immobilized on the stationary phase is maintained at a constant temperature value (e.g., the optimal temperature) during at least a portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is maintained at a selected temperature value (e.g., the optimal temperature) ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C during at least a portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is maintained at 37°C ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C during at least a portion of the stimulation. In some aspects, stimulation of cells (e.g., lymphocytes such as T cells) at an optimal temperature (e.g., 37°C or 37°C ± about 5°C) facilitates cell activation and downstream processing of the cells, e.g., detachment or elution of the cells from the stationary phase and / or genetic modification of the cells. In some aspects, stimulation of cells (e.g., lymphocytes such as T cells) at an optimal temperature (e.g., 37°C or 37°C ± about 5°C) protects or maintains the health of the cells during on-column stimulation.

[0182] In some aspects, stimulation of cells (e.g., lymphocytes such as T cells) in the presence of air (e.g., air) within the column at an optimal temperature (e.g., 37°C or 37°C ± about 5°C) facilitates activation of the cells and downstream processing of the cells, e.g., detachment or elution of the cells from the stationary phase and / or genetic modification of the cells.

[0183] 1A-1B illustrate an exemplary column chromatography housing assembly. In some aspects, housing assembly 1 includes an inlet housing member 2 and an outlet housing member 3, where at least the inlet housing member and the outlet housing member form an internal cavity configured to accommodate a stationary phase, such as a chromatographic resin 4. In some aspects, the housing assembly further includes a temperature control member configured to provide heat to the stationary phase in the internal cavity, e.g., a temperature control member including a heating coil 5. In some aspects, the housing assembly further includes a connector, e.g., a gas exchange connector 6, configured to operably connect the internal cavity to a gas source, thereby enabling or achieving gas introduction into the internal cavity. In some embodiments, the connector is disposed on the inlet housing member. In some embodiments, the connector is disposed on the outlet housing member. In some embodiments, both the inlet housing member and the outlet housing member have a connector disposed thereon. For example, as shown in FIG. 1A, both the inlet housing member 2 and the outlet housing member 3 have a gas exchange connector 6 disposed thereon.

[0184] In some embodiments, the housing assembly further comprises a sidewall member. For example, as shown in Figure 1A, inlet housing member 2, outlet housing member 3 and sidewall member 7 collectively form an interior cavity.

[0185] In some embodiments, the connector is disposed on the inlet housing member. In some embodiments, the connector is disposed on the outlet housing member. In some embodiments, the connector is disposed on the sidewall member. In some embodiments, both the inlet housing member and the sidewall member have connectors disposed thereon. In some embodiments, both the outlet housing member and the sidewall member have connectors disposed thereon. In some embodiments, each of the inlet housing member, the outlet housing member, and the sidewall member has a connector disposed thereon. In some embodiments, the inlet housing member has at least two connectors disposed thereon. In some embodiments, the outlet housing member has at least two connectors disposed thereon. In some embodiments, the sidewall member has at least two connectors disposed thereon.

[0186] In some embodiments, connectors are formed between any two or all three of the inlet housing member, the outlet housing member, and the sidewall member. In some aspects, connectors are formed between the inlet housing member and the outlet housing member. In some aspects, connectors are formed between the inlet housing member and the sidewall member. In some aspects, connectors are formed between the outlet housing member and the sidewall member. In some aspects, at least one connector is formed between the inlet housing member and the sidewall member and at least one connector is formed between the outlet housing member and the sidewall member.

[0187] In any of the foregoing embodiments, the housing assembly can include multiple connectors, e.g., gas exchange connectors 6, configured to operably connect the internal cavity to a gas source, thereby enabling or achieving the introduction of gas into the internal cavity. In some embodiments, at least one of the connectors is operably connected to a gas source (directly or indirectly via tubing, optionally including one or more filters and / or one or more valves), while at least one other connector is configured to vent.

[0188] In any of the above embodiments, the connector can be a tie-type connector, a threaded connector, a Luer connector (e.g., a Luer lock connector or a Luer slip connector), a barbed connector, or any combination thereof. In any of the above embodiments, the connector can include a male fitting or a female fitting. In any of the above embodiments, the connector can be configured to sealingly engage tubing in fluid communication with a gas source. In any of the above embodiments, the connector can include one or more valves. In any of the above embodiments, the connector can be operatively connected to tubing including one or more valves. In any of the above embodiments, the connector can include one or more filters. In any of the above embodiments, the connector can be operatively connected to tubing including one or more filters. In any of the above embodiments, the one or more filters can be gas filters, e.g., air filters. In any of the above embodiments, the one or more filters can be sterile filters and / or sterilizing filters for sterile filtration.

[0189] In some aspects, the housing assembly includes an inlet housing member including a top cover. In some embodiments, the top cover is removably attached to the inlet housing member or the side wall member. In some embodiments, the top cover is integrally formed with the inlet housing member or the side wall member. In some embodiments, the connector is disposed on the top cover.

[0190] In any of the above embodiments, the inlet housing member can include one or more inlets operatively connected to the internal cavity to allow introduction of an input composition into the internal cavity. For example, as shown in FIG. 1A, the inlet housing member 2 includes an inlet, e.g., a tubing set connector 8, disposed on the top cover. In some embodiments, the connector and the one or more inlets are disposed in different locations on the top cover, as shown in FIGS. 1A (bottom panel) and 1B. In some embodiments, the connector and the one or more inlets are disposed in the same location on the top cover. For example, the one or more inlets are configured to operatively connect the internal cavity to a gas source, thereby enabling or achieving introduction of gas into the internal cavity, and are simultaneously configured to operatively connect to the internal cavity to allow introduction of an input composition into the internal cavity. The one or more inlets can be controllably opened and closed to introduce gas at certain times during chromatography and to introduce an input composition at other times during chromatography.

[0191] In some embodiments, the fluid path through the one or more inlets is at an angle of about 90 degrees relative to the top cover, while the fluid path through the connector is at an angle of about 45 degrees relative to the top cover.

[0192] In any of the above aspects, the outlet housing member can include a bottom cover of the housing assembly. In some aspects, the bottom cover is removably attached to the outlet housing member or the sidewall member. In other aspects, the bottom cover is integrally formed with the outlet housing member or the sidewall member.

[0193] In any of the above embodiments, the outlet housing member can include one or more outlets operatively connected to the internal cavity to allow or effect the discharge of the output composition from the internal cavity. In some aspects, the one or more outlets are disposed in the bottom cover. In some embodiments, the connector and the one or more outlets are disposed in different locations on the bottom cover, as shown, for example, in FIG. 1A (bottom panel). In some embodiments, the connector and the one or more outlets are disposed in the same location on the bottom cover. For example, the one or more outlets are configured to operatively connect the internal cavity to a gas source, thereby allowing or effecting the introduction of gas into the internal cavity, and are also configured to operatively connect to the internal cavity to allow or effect the discharge of the output composition from the internal cavity. The one or more outlets can be controllably opened and closed to allow the introduction of gas at certain times during chromatography, and can be controllably opened and closed to allow the discharge of the output composition from the internal cavity at other times during chromatography. In some embodiments, the fluid path through the one or more outlets forms an angle of about 90 degrees with respect to the bottom cover.

[0194] In any of the above embodiments, the gas source can be or include a gas reservoir or the external environment. In any of the above embodiments, the gas in the gas source can be sterile. In any of the above embodiments, the gas can be or include air.

[0195] In any of the preceding embodiments, the housing assembly can further include tubing operatively connected to a gas source. In some embodiments, the tubing is configured to sterilely connect the interior cavity to the gas source. In any of the preceding embodiments, the tubing can include one or more valves. In any of the preceding embodiments, the tubing can include one or more filters.

[0196] In any of the above embodiments, the housing assembly can further include one or more porous members, e.g., a cell strainer or cell sieve. For example, as shown in FIG. 1A (top panel), housing assembly 1 includes a woven polyester mesh 9. In some embodiments, the housing assembly includes a first porous member, e.g., a woven polyester mesh 9, between inlet housing member 2 and sidewall member 7, configured to separate the stationary phase from the inlet of the internal cavity. In some embodiments, the housing assembly further includes a second porous member, e.g., a woven polyester mesh 9, between outlet housing member 3 and sidewall member 7, configured to separate the stationary phase from the outlet of the internal cavity.

[0197] In any of the above embodiments, the one or more porous members can have an average pore size of about 20 μm. In any of the above embodiments, the one or more porous members can include a mesh having a mesh size of about 20 μm.

[0198] In any of the above embodiments, the temperature control member can be configured to adjust or maintain the temperature of the stationary phase in the internal cavity. In some aspects, the temperature control member is configured to heat the stationary phase in the internal cavity from a starting temperature (e.g., room temperature) to a target temperature of about 35°C to about 39°C (e.g., 37°C or about 37°C) during a chromatography run. In some aspects, the temperature control member is further configured to maintain the stationary phase at the target temperature.

[0199] In any of the preceding embodiments, the housing assembly can further include a temperature sensor configured to measure the temperature of the stationary phase in the internal cavity. In one aspect, the temperature sensor is configured to couple to a monitoring / display unit. In some embodiments, the temperature sensor is configured to electrically connect to a power source. In some embodiments, the power source is external to the housing assembly. In some embodiments, the housing assembly further includes a power source.

[0200] In any of the above aspects, the temperature control member can include a heat source. Alternatively, in any of the above aspects, the temperature control member can be configured to operatively connect to a heat source external to the housing assembly.

[0201] In some embodiments, the temperature control member comprises a heating element. In some embodiments, the heating element is configured to heat the stationary phase evenly.

[0202] In any of the above embodiments, the temperature control member can include a heating element selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In one aspect, the heating element is an electric heating element. In some embodiments, the electric heating element includes a metal plate, a metal rod, a metal wire, or a combination thereof. In one aspect, the heating element is an electromagnetic induction heating element. In some embodiments, the electromagnetic induction heating element includes an induction heating coil surrounding a magnetizable core configured to provide heat to a stationary phase in the internal cavity. In one aspect, the heating element is a non-electric heating element.

[0203] In some embodiments, the non-electrical heating element includes a heating flow path including an inlet and an outlet for a heated fluid, such as a heated liquid or gas. In some embodiments, the heating flow path is a heating coil and the heated fluid is heated water. For example, as shown in FIG. 1A, the housing assembly includes a heating coil inlet 10 and a heating coil outlet 11. In some embodiments, the heated water inlet is configured to connect to an external reservoir of heated water.

[0204] In some embodiments, the heating element is an electric heating element. In some embodiments, the electric heating element is configured to be electrically connected to a power source. In some embodiments, the power source is external to the housing assembly. In some embodiments, the housing assembly further comprises a power source.

[0205] In some embodiments, the electric heating element comprises a metal plate. In some embodiments, the metal plate is made at least in part of a thermally conductive metal, such as aluminum or copper. In some embodiments, the metal plate is made at least in part of aluminum (e.g., entirely of aluminum). In some embodiments, the electric heating element further comprises an electrical isolation layer. In some embodiments, the electrical isolation layer is between at least a portion of the metal plate and at least a portion of another component of the electric heating element. In some embodiments, the electrical isolation layer lines at least a portion of one side of the metal plate (e.g., lines the entire one side of the metal plate).

[0206] In some embodiments, at least a portion of the heating element contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, at least a portion of the heating element contacts, e.g., directly contacts, at least a portion of the sidewall member. In some embodiments, at least a portion of the heating element contacts, e.g., directly contacts, at least a portion of the inlet housing member. In some embodiments, at least a portion of the heating element contacts, e.g., directly contacts, at least a portion of the outlet housing member.

[0207] In some embodiments, the heating element contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or the sidewall member. In some embodiments, the heating element contacts, e.g., directly contacts, at least a portion of the sidewall member. In some embodiments, the heating element contacts, e.g., directly contacts, the sidewall member.

[0208] In some embodiments, at least a portion of the heating element is not in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the heating element is not in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the heating element is not in contact with the inlet housing member, the outlet housing member, or the sidewall member.

[0209] In any of the above embodiments, the heating element can be disposed along and / or around the central axis of the internal cavity. In some aspects, the heating element is disposed inside the internal cavity, outside the internal cavity, or partially inside and partially outside the internal cavity. In some aspects, the heating element is disposed inside the sidewall member, outside the sidewall member, or partially inside and partially outside the sidewall member.

[0210] In some embodiments, a heating element is disposed inside the internal cavity. In some embodiments, the heating element comprises a non-electric heating element disposed inside the internal cavity. In some embodiments, the heating element comprises a heating channel disposed inside the internal cavity. In some embodiments, the heating channel comprises an inlet and an outlet for a heated fluid, for example, heated water. In some embodiments, the heated water inlet is configured to connect to an external reservoir of heated water.

[0211] In some embodiments, the heating element is disposed outside the internal cavity. In some embodiments, the heating element is disposed outside the sidewall member. In some embodiments, the heating element surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the heating element surrounds (e.g., completely surrounds) the sidewall member. In some embodiments, the heating element surrounds at least a portion of the inlet housing member. In some embodiments, at least a portion of one of the one or more inlets of the inlet housing member, e.g., one of the one or more inlets operatively connected to the internal cavity to allow intake of an input composition into the internal cavity, is exposed from the heating element. In some embodiments, at least a portion of one of the one or more inlets of the inlet housing member operatively connected to the internal cavity to allow intake of an input composition into the internal cavity is exposed from the heating element. In some embodiments, at least a portion of one of the one or more inlets of the inlet housing member is outside the heating element. In some embodiments, the heating element surrounds at least a portion of the outlet housing member. In some embodiments, at least a portion of one of the one or more outlets of the outlet housing member, e.g., one of the one or more outlets operatively connected to the internal cavity to enable or achieve discharge of the output composition from the internal cavity, is exposed from the heating element. In some embodiments, at least a portion of one of the one or more outlets operatively connected to the internal cavity to enable or achieve discharge of the output composition from the internal cavity, is exposed from the heating element. In some embodiments, at least a portion of one of the one or more outlets of the outlet housing member is external to the heating element.

[0212] In some embodiments, the heating element comprises a heating channel that surrounds at least a portion of the inlet housing member, the outlet housing member, and / or the sidewall member.

[0213] In any of the preceding embodiments, the heating element can include a coil surrounding the inlet housing member, the outlet housing member, and / or the sidewall member. In any of the preceding embodiments, the heating element can include a heating channel surrounding the inlet housing member, the outlet housing member, and / or the sidewall member. In any of the preceding embodiments, the heating element can include a heating channel surrounding the sidewall member.

[0214] In some embodiments, the heating element surrounds at least a portion of the sidewall member, at least a portion of the outlet housing member, and / or at least a portion of the inlet housing member, and the housing assembly further includes an insulating layer between the heating element and at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the insulating layer surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the insulating layer surrounds at least a portion of the sidewall member, e.g., completely surrounds the sidewall member. In some embodiments, the insulating layer is a solid layer. In some embodiments, the insulating layer is a liquid layer. In some embodiments, the insulating layer is a gas layer. In some embodiments, the insulating layer is an air layer.

[0215] In some embodiments, the temperature control member comprises multiple heating elements. In some embodiments, the temperature control member comprises 2 to 10 or about 2 to 10 heating elements, 2 to 8 or about 2 to 8 heating elements, 2 to 6 or about 2 to 6 heating elements, or 2 to 4 or about 2 to 4 heating elements, inclusive. In some embodiments, the temperature control member comprises two heating elements. In some embodiments, the temperature control member comprises three heating elements. In some embodiments, the temperature control member comprises four heating elements.

[0216] In some embodiments, the plurality of heating elements are configured to heat the stationary phase evenly. In some embodiments, the plurality of heating elements are positioned to heat the stationary phase evenly.

[0217] In some embodiments, each of the plurality of heating elements is selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In some embodiments, the plurality of heating elements are identical. In some embodiments, the plurality of heating elements is a combination of different heating elements.

[0218] In some embodiments, the plurality of heating elements comprises a plurality of non-electric heating elements. In some embodiments, the plurality of heating elements comprises a plurality of heating channels. In some embodiments, each of the plurality of heating channels has an inlet and an outlet for a heated fluid, e.g., heated water. In some embodiments, at least two of the plurality of heating channels are fluidly connected to each other. In some embodiments, the plurality of heating channels are fluidly connected to each other. In some embodiments, the inlet of at least one of the plurality of heating channels is configured to connect to an external reservoir of heated fluid, e.g., heated water. In some embodiments, the inlet of each of the plurality of heating channels is configured to connect to an external reservoir of heated fluid.

[0219] In some embodiments, the plurality of heating elements comprises a plurality of electric heating elements, e.g., an electric heating element comprising a metal plate. In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to each other. In some embodiments, the plurality of electric heating elements are electrically coupled to each other. In some embodiments, at least one of the plurality of electric heating elements is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly. In some embodiments, each of the plurality of electric heating elements is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0220] In some embodiments, at least a portion of at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, at least a portion of at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the inlet housing member. In some embodiments, at least a portion of at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the outlet housing member. In some embodiments, at least a portion of at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the sidewall member.

[0221] In some embodiments, at least a portion of at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, at least one of the plurality of heating elements contacts, e.g., directly contacts, at least a portion of the sidewall member. In some embodiments, at least one of the plurality of heating elements contacts, e.g., directly contacts, the sidewall member.

[0222] In some embodiments, at least a portion of at least one of the plurality of heating elements is not in contact with at least a portion of the inlet housing member, at least a portion of the outlet housing member, or at least a portion of the sidewall member. In some embodiments, at least a portion of at least one of the plurality of heating elements is not in contact with the inlet housing member, the outlet housing member, or the sidewall member. In some embodiments, at least one of the plurality of heating elements is not in contact with the inlet housing member, the outlet housing member, or the sidewall member. In some embodiments, the plurality of heating elements is not in contact with the inlet housing member, the outlet housing member, or the sidewall member.

[0223] In some embodiments, at least one of the plurality of heating elements is disposed along and / or around the central axis of the internal cavity. In some embodiments, at least one of the plurality of heating elements is disposed inside the internal cavity, outside the internal cavity, or partially inside and partially outside the internal cavity. In some embodiments, at least one of the plurality of heating elements is disposed inside a sidewall member, outside a sidewall member, or partially inside and partially outside a sidewall member. In some embodiments, at least one of the plurality of heating elements is disposed inside the internal cavity and at least one of the plurality of heating elements is disposed outside the internal cavity. In some embodiments, the plurality of heating elements is disposed inside the internal cavity. In some embodiments, the plurality of heating elements is disposed outside the internal cavity. In some embodiments, the plurality of heating elements is disposed outside the sidewall member.

[0224] In some embodiments, at least one of the multiple heating elements is disposed outside the internal cavity. In some embodiments, at least one of the multiple heating elements surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, at least one of the multiple heating elements surrounds at least a portion of the sidewall member, e.g., completely surrounds the sidewall member. In some embodiments, the multiple heating elements surround at least a portion of the sidewall member, e.g., completely surrounds the sidewall member. In some embodiments, at least one of the multiple heating elements surrounds at least a portion of the inlet housing member. In some embodiments, at least a portion of one or more inlets of the inlet housing member is exposed from the multiple heating elements. In some embodiments, at least a portion of one or more inlets of the inlet housing member is outside the multiple heating elements. In some embodiments, at least one of the multiple heating elements surrounds at least a portion of the outlet housing member. In some embodiments, the multiple heating elements surround at least a portion of the outlet housing member. In some embodiments, at least a portion of the one or more outlets of the outlet housing member are exposed from the plurality of heating elements, hi some embodiments, at least a portion of the one or more outlets of the outlet housing member are exterior to the plurality of heating elements.

[0225] In some embodiments, the plurality of heating elements are evenly or approximately evenly distributed around the sidewall member. In some embodiments, the plurality of heating elements are evenly or approximately evenly distributed around the periphery of the sidewall member.

[0226] In some embodiments, at least one of the plurality of heating elements surrounds at least a portion of the sidewall member, at least a portion of the outlet housing member, and / or at least a portion of the inlet housing member, and the housing assembly further includes an insulating layer between at least one of the plurality of heating elements and at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the insulating layer surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the insulating layer surrounds at least a portion of the sidewall member, e.g., completely surrounds the sidewall member. In some embodiments, the insulating layer is a liquid layer. In some embodiments, the insulating layer is a gas layer. In some embodiments, the insulating layer is an air layer.

[0227] In some embodiments, the heating element is disposed on the exterior of the sidewall member, and the housing assembly further comprises a jacket member (also referred to herein as a jacket) comprising the heating element. In some embodiments, the jacket member comprises a temperature control member comprising the heating element disposed on the exterior of the sidewall member. In some embodiments, the jacket member is any of those described in Section IC.

[0228] In some embodiments, at least one of the plurality of heating elements is disposed outside the sidewall member, and the housing assembly further comprises a jacket member comprising at least one of the plurality of heating elements. In some embodiments, the jacket member comprises a temperature control member comprising at least one of the plurality of heating elements. In some embodiments, the plurality of heating elements is disposed outside the sidewall member, and the housing assembly further comprises a jacket member comprising the plurality of heating elements. In some embodiments, the jacket member comprises a temperature control member comprising the plurality of heating elements.

[0229] In some embodiments, the jacket member is configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the jacket member surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0230] In some embodiments, the jacket members are removably connected together and surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member, hi some embodiments, the jacket members are not removably connected to each other.

[0231] In some embodiments, the jacket member is configured to surround at least a portion of the sidewall member. In some embodiments, the jacket member is configured to completely surround the sidewall member. In some embodiments, the jacket member surrounds at least a portion of the sidewall member, e.g., completely surrounds the sidewall member. In some embodiments, the jacket member completely surrounds the sidewall member. In some embodiments, the jacket member surrounds at least a portion of the inlet housing member. In some embodiments, one or more inlets of the inlet housing member are exposed by the jacket member. In some embodiments, one or more inlets of the inlet housing member operatively connected to the internal cavity to allow for the intake of an input composition into the internal cavity are exposed by the jacket member. In some embodiments, one or more inlets of the inlet housing member are exterior to the jacket member. In some embodiments, the jacket member surrounds at least a portion of the outlet housing member. In some embodiments, one or more outlets of the outlet housing member operatively connected to the internal cavity to allow or achieve the discharge of an output composition from the internal cavity are exposed by the jacket member. In some embodiments, one or more outlets of the outlet housing member are external to the jacket member.

[0232] In some embodiments, the jacket member contacts, e.g., directly contacts, at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the jacket member contacts, e.g., directly contacts, at least a portion of the sidewall member. In some embodiments, the jacket member contacts, e.g., directly contacts, the sidewall member.

[0233] In some embodiments, at least a portion of the jacket member does not contact at least a portion of the inlet housing member, at least a portion of the outlet housing member, or at least a portion of the sidewall member. In some embodiments, at least a portion of the jacket member does not contact the inlet housing member, the outlet housing member, or the sidewall member. In some embodiments, the jacket member does not contact the inlet housing member, the outlet housing member, or the sidewall member.

[0234] In some embodiments, the jacket member includes a non-electric heating element, e.g., a heating flow path including an inlet and an outlet for a heated fluid. In some embodiments, the jacket member includes multiple non-electric heating elements. In some embodiments, the jacket member includes at least one opening for an inlet for a heated fluid. In some embodiments, the jacket member includes at least one opening for an outlet for a heated fluid. In some embodiments, the jacket member includes at least two openings for one or more inlets for a heated fluid. In some embodiments, the jacket member includes at least two openings for one or more outlets for a heated fluid, e.g., heated water. In some embodiments, the jacket member is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0235] In some embodiments, the jacket member comprises an electric heating element, e.g., an electric heating element comprising a metal plate. In some embodiments, the jacket member comprises a plurality of electric heating elements. In some embodiments, the jacket member is arranged such that the electric heating element or plurality of electric heating elements are configured to electrically connect to a power source.

[0236] In some embodiments, the jacket member includes at least one temperature sensor configured to measure the temperature of the stationary phase in the internal cavity, hi some embodiments, the temperature sensor is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0237] In some embodiments, the jacket member comprises one or more jacket components. In some embodiments, the one or more jacket components are configured to collectively form the jacket member. In some embodiments, the one or more jacket components are configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member. In some embodiments, the one or more jacket components are configured to be removably connected together and to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

[0238] In some embodiments, the one or more jacket components are configured to surround at least a portion of the sidewall member, for example, to completely surround the sidewall member. In some embodiments, the one or more jacket components are configured to completely surround the sidewall member. In some embodiments, the one or more jacket components are configured to surround at least a portion of the inlet housing member. In some embodiments, one or more inlets of the inlet housing member are exposed from the one or more jacket components. In some embodiments, one or more inlets of the inlet housing member are exterior to the one or more jacket components. In some embodiments, the one or more jacket components are configured to surround at least a portion of the outlet housing member. In some embodiments, one or more outlets of the outlet housing member are exposed from the one or more jacket components. In some embodiments, one or more outlets of the outlet housing member are exterior to the one or more jacket components.

[0239] In some embodiments, the jacket member includes two or more jacket parts, e.g., 2 to 10 or about 2 to 10 jacket parts, 2 to 8 or about 2 to 8 jacket parts, 2 to 6 or about 2 to 6 jacket parts, or 2 to 4 or about 2 to 4 jacket parts, inclusive. In some embodiments, the jacket member includes two jacket parts. In some embodiments, the jacket member includes three jacket parts. In some embodiments, the jacket member includes four jacket parts.

[0240] In some embodiments, at least two of the two or more jacket components each comprise a heating element. In some embodiments, the two or more jacket components each comprise a heating element.

[0241] In some embodiments, at least two of the two or more jacket components each comprise a temperature sensor. In some embodiments, the two or more jacket components each comprise a temperature sensor.

[0242] In some embodiments, at least two of the two or more jacket components each include a non-electric heating element. In some embodiments, at least two of the two or more jacket components each include a heated flow channel with an inlet and an outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include a heated flow channel with an inlet and an outlet for a heated fluid, e.g., heated water.

[0243] In some embodiments, at least two heating channels of the two or more jacket components are fluidly connected to one another. In some embodiments, the heating channels of the two or more jacket components are fluidly connected to one another.

[0244] In some embodiments, at least one of the two or more jacket components includes an opening for an inlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for an inlet for a fluid, e.g., heated water.

[0245] In some embodiments, at least one inlet of the heating channels of the two or more jacket components is configured to connect to an external reservoir of heated fluid. In some embodiments, each inlet of the heating channels of the two or more jacket components is configured to connect to an external reservoir of heated fluid.

[0246] In some embodiments, at least one of the two or more jacket components includes an opening for an outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for an outlet for a fluid, e.g., heated water.

[0247] Figures 25-28 are schematic diagrams of exemplary housing assemblies for column chromatography. The exemplary housing assembly 1 shown in Figure 25 includes an inlet housing member 2, an outlet housing member 3, and a sidewall member 7 that form an internal cavity configured to accommodate a stationary phase. The housing assembly 1 also includes a gas supply connector (not shown) for a threaded air filter and a temperature control member including a heating coil 5, as shown in Figures 26A-26C. The heating coil 5 is contained within a jacket member made of two jacket parts 12. The jacket parts 12 are configured to collectively completely surround the sidewall member 7 and to surround at least a portion of each of the inlet housing member 2 and the outlet housing member 3. Each jacket part 12 includes an inlet groove 13 so that the jacket part exposes the inlet of the inlet housing member 2. Each jacket part 12 also includes an outlet groove 14 so that the jacket part exposes the outlet of the outlet housing member 3.

[0248] 26A-26C are interior, side, and exterior views of jacket components 12. As shown in FIGS. 26A-26C, each jacket component 12 includes a heating coil 5 for a heated fluid, such as heated water. The two heating coils 5 of a jacket component are configured to collectively completely surround the sidewall member 7 and to surround at least a portion of each of the inlet housing member 2 and the outlet housing member 3. Each jacket component 12 also includes openings for a heating coil inlet 10 and a heating coil outlet 11 of the heating coil. As shown in FIG. 27, the heating coil inlet 10 is aligned with the inlet of the inlet housing member 2. As shown in FIG. 28, the heating coil outlet 1 is aligned with the outlet of the outlet housing member 3.

[0249] In some embodiments, at least two of the two or more jacket components each include an electric heating element, e.g., an electric heating element including a metal plate. In some embodiments, at least two of the two or more jacket components each include an electric heating element. In some embodiments, the two or more jacket components each include an electric heating element.

[0250] In some embodiments, the electric heating elements of at least two of the two or more jacket components are electrically coupled to each other. In some embodiments, the electric heating elements of the two or more jacket components are electrically coupled to each other.

[0251] In some embodiments, at least one of the two or more jacket components is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly. In some embodiments, each of the two or more jacket components is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0252] In some embodiments, at least one electric heating element of at least two of the two or more jacket components is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly. In some embodiments, each electric heating element of the two or more jacket components is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0253] 29-31 are schematic diagrams of an exemplary housing assembly for column chromatography. The exemplary housing assembly 1 shown in FIG. 29 includes an inlet housing member 2, an outlet housing member 3, and a sidewall member 7 that form an internal cavity configured to accommodate a stationary phase. The housing assembly 1 also includes a gas supply connector (not shown) for a threaded air filter and a temperature control member including an electric heating element 17 comprising a metal plate. The electric heating element 17 is part of a jacket member made of three jacket parts 12. The jacket parts 12 are configured to collectively completely surround the sidewall member 7 and to surround at least a portion of each of the inlet housing member 2 and the outlet housing member 3. Each jacket part 12 includes an inlet groove 13 so that the jacket member exposes the inlet of the inlet housing member 2. Each jacket part 12 also includes an outlet groove 14 so that the jacket member exposes the outlet of the outlet housing member 3.

[0254] FIGS. 30A-30C show three views of the jacket component 12. Each jacket component 12 includes an electric heating element 17 and a temperature sensor 18. Each electric heating element 17 is configured to electrically connect to a power source via a heating element electrical connection point 16, and each temperature sensor is configured to electrically connect to a power source via a temperature sensor electrical connection point 20. As shown in FIG. 30D, the electric heating element 17 also includes an electrical isolation layer 19 and an aluminum profile 21. Each electric heating element 17 is attached to a mounting location 15 of the jacket component 12. The electric heating elements 17 and jacket component 12 are configured so that the electric heating elements 17 are evenly distributed around the periphery of the sidewall member 7. As shown in FIG. 29, the heating element electrical connection points 16 and the temperature sensor electrical connection points 20 are exposed on the same side of the jacket component as the outlet of the outlet housing member 3.

[0255] In one aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element disposed along and / or around a central axis of the internal cavity, the heating element configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of a sterile gas into the internal cavity.

[0256] In one aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase, the temperature control member including a heating element disposed along and / or about a central axis of the internal cavity, the heating element configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of a sterile gas into the internal cavity.

[0257] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element comprising a metal plate configured to impart heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of a sterile gas into the internal cavity.

[0258] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, the inlet housing member, the outlet housing member, and the sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to regulate or maintain the temperature of the stationary phase, the temperature control member including two heating coils configured to apply heat to the stationary phase; a jacket member including the temperature control member including the two heating coils, the jacket members removably connected together and surrounding at least a portion of the inlet housing member, the outlet housing member, and the sidewall member, the two heating coils completely surrounding the sidewall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0259] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, the inlet housing member, the outlet housing member, and the sidewall member forming an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member configured to regulate or maintain the temperature of the stationary phase, the temperature control member including three electric heating elements, each including a metal plate, configured to apply heat to the stationary phase; a jacket member including the temperature control member including the three electric heating elements, the jacket members being detachably connected together and surrounding at least a portion of the inlet housing member, the outlet housing member, and the sidewall member, and two heating coils completely surrounding the sidewall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0260] In yet another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element including a heating coil configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas source, thereby enabling or achieving the introduction of sterile gas into the internal cavity. In some embodiments, the heating coil includes an inlet and an outlet for heated water. In some aspects, the heating coil surrounds the inlet housing member, the outlet housing member, and the sidewall member.

[0261] In one aspect, disclosed herein is a housing assembly for column chromatography including an inlet housing member, an outlet housing member, and a sidewall member, wherein the inlet housing member, the outlet housing member, and the sidewall member form an internal cavity configured to accommodate a stationary phase for column chromatography; a temperature control member including a heating element configured to provide heat to the stationary phase in the internal cavity; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby enabling or achieving the introduction of sterile gas into the internal cavity.

[0262] In any of the preceding aspects, the gas filter can be an air filter and the sterile gas can be sterile air. In any of the preceding aspects, the housing assembly can further include a gas filter.

[0263] Also disclosed herein is a housing assembly set including a plurality of housing assemblies disclosed herein. The housing assembly set can include at least two of the plurality of housing assemblies arranged in series. The housing assembly set can include at least two of the plurality of housing assemblies arranged in parallel.

[0264] Also disclosed herein are chromatography systems including any of the housing assemblies disclosed herein and at least one additional chromatography column. In some embodiments, the at least one additional chromatography column does not include a temperature control member. In some embodiments, the at least one additional chromatography column does not include a connector configured to operably connect an internal cavity of the at least one additional chromatography column to a gas source.

[0265] B. Chromatography Kits, Columns, and Column Sets In some embodiments, a chromatography kit is also disclosed herein, comprising a housing assembly or housing assembly set disclosed herein and a stationary phase for chromatography. In some embodiments, the housing assembly or housing assembly set is any of those described in Section IA. In some embodiments, the chromatography kit further comprises one or more stimuli or reagents. In some embodiments, the one or more stimuli or reagents are any of those described in Section II-B-1 or II-B-2.

[0266] In some embodiments, also disclosed herein is a chromatography column or a chromatography column set comprising a housing assembly or a housing assembly set disclosed herein and a stationary phase for column chromatography in one or more internal cavities of the housing assembly. In some embodiments, the housing assembly or housing assembly set is any of those described in Section IA.

[0267] In some embodiments, a chromatography column is also disclosed herein, comprising a jacket member and a chromatography column. In some embodiments, the interior cavity of the chromatography column comprises a stationary phase for chromatography. In some embodiments, the jacket member is any of those described in Section IC.

[0268] In some embodiments, a chromatography column set is also disclosed herein, comprising at least one jacket member and a plurality of chromatography columns. In some embodiments, the jacket member is any of those described in Section IC. In some embodiments, the interior cavity of each of the plurality of chromatography columns comprises a stationary phase for chromatography. In some embodiments, the plurality of chromatography columns are arranged in series. In some embodiments, the plurality of chromatography columns are arranged in parallel. In some embodiments, the plurality of chromatography columns are operatively connected.

[0269] In some embodiments, the plurality of chromatography columns comprises a first chromatography column. In some embodiments, the plurality of chromatography columns comprises a second chromatography column. In some embodiments, the at least one jacket member is configured to surround the second chromatography column.

[0270] In any of the above embodiments, the stationary phase can comprise a gel filtration matrix and / or an affinity chromatography matrix. The stationary phase can comprise a non-magnetic material, a non-ferromagnetic material, or a paramagnetic material. In another aspect, the stationary phase is selected from the group consisting of cellulose membranes, plastic membranes, polysaccharide gels, polyacrylamide gels, agarose gels, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, poly(N-isopropylacrylamide)-grafted silica, styrene-divinylbenzene gels, copolymers of acrylates or acrylamides and diols, copolymers of polysaccharides and N,N'-methylenebisacrylamide, and combinations thereof. The stationary phase can comprise or be a monolithic matrix, a particulate matrix, and / or a planar matrix.

[0271] In any of the above embodiments, the particulate matrix can have an average particle size of about 5 μm to about 200 μm, about 5 μm to about 600 μm, or about 5 μm to about 1500 μm. In any of the above embodiments, the stationary phase can have an average pore size of about 1 nm to about 500 nm.

[0272] In any of the above embodiments, the stationary phase can include any of the agents described in Section II-B-1 immobilized thereon. In some embodiments, the agent is directly immobilized on the stationary phase. In some embodiments, the agent is indirectly immobilized on the stationary phase. In some embodiments, the agent is irreversibly immobilized on the stationary phase. In some embodiments, the agent is reversibly immobilized on the stationary phase. In some embodiments, the agent is reversibly immobilized on the stationary phase via a mutein of streptavidin that reversibly binds to the streptavidin-binding peptide. In some embodiments, the streptavidin mutein and / or streptavidin-binding peptide is any of those described in Section II-B-2.

[0273] In any of the above embodiments, the stationary phase can include a selection agent immobilized thereon. In some aspects, the selection agent is capable of specifically binding to a selection marker on the surface of one or more cells. In some embodiments, the one or more cells are immune cells. In some aspects, the one or more cells are T cells.

[0274] Also disclosed herein is a device comprising a housing assembly, a housing assembly set, or a chromatography kit, a chromatography column, or a chromatography column set, further comprising an input composition reservoir operatively connected to the internal cavity via an inlet in the inlet housing member. In some embodiments, the input composition comprises blood or a blood-derived sample, or is blood or a blood-derived sample. In some embodiments, the input composition comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product, or is a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. In some embodiments, the apheresis product or leukocyte apheresis product is freshly isolated from a subject or thawed from a cryopreserved apheresis product or leukocyte apheresis product. In some embodiments, the device further includes an output composition reservoir operatively connected to the internal cavity via the outlet of the outlet housing member. In some aspects, the output composition includes or is enriched T cells. In another aspect, the enriched T cells have been stimulated during chromatography on the chromatography column. In any of the above embodiments, the device can be a closed system or a sterile system. An exemplary device including an exemplary housing assembly 1 is shown in FIG. 1B.

[0275] Also disclosed herein is a method for preparing a chromatography column or a chromatography column set, comprising introducing a stationary phase into a housing assembly or a housing assembly set disclosed herein. Additionally, disclosed herein is a method for preparing a chromatography column or a chromatography column set, comprising introducing a stationary phase of a chromatography kit into a housing assembly or a housing assembly set of the chromatography kit.

[0276] C. Chromatography jacketing The devices provided herein also include jacket members for column chromatography. In some aspects, the provided jacket members are devices that enable improved methods involving the isolation, processing, or manipulation of target cells immobilized on the stationary phase of a chromatography column, such as improved methods for on-column selection and / or stimulation of target cells. In some aspects, the provided jacket members allow for temperature regulation, e.g., heating, of the immobilized cells. In some aspects, the provided jacket members allow for the temperature of the immobilized cells to be maintained, for example, at or about 37°C, or 37°C ± about 5°C. In some aspects, regulating and maintaining the temperature of the cells using the provided devices improves on-column manipulation, e.g., stimulation, of the immobilized cells, for example, by improving the overall health, fitness, or condition of the immobilized cells during on-column manipulation.

[0277] In one aspect, the jacket member includes one or more jacket components configured to surround at least a portion of the chromatography column. In some aspects, the chromatography column is configured to accommodate a stationary phase. In some aspects, the chromatography column includes a stationary phase. In some aspects, the jacket member further includes one or more heating elements. In some aspects, the one or more heating elements are configured to provide heat to the stationary phase. In some embodiments, the one or more heating elements are configured to be part of a temperature control member. In some aspects, the temperature control member is configured to regulate or maintain the temperature of the stationary phase. In some embodiments, the one or more heating elements are any of those described in Section IA. In some embodiments, the temperature control member is any of those described in Section IA.

[0278] In some aspects, the one or more jacket components are configured to be removably connected together to surround at least a portion of the chromatography column, hi other embodiments, the one or more jacket components are configured not to be removably connected to each other.

[0279] In some embodiments, the jacket member has a volume of 1 to 40 mL or about 1 to 40 mL, e.g., 1 to 35 mL, 1 to 30 mL, 1 to 25 mL, 1 to 20 mL, 1 to 15 mL, 1 to 10 mL, 1 to 5 mL, 5 to 40 mL, 5 to 35 mL, 5 to 30 mL, 5 to 25 mL, 5 to 20 mL, 5 to 15 mL, 5 to 10 mL, 10 to 40 mL, 10 to 35 mL, 10 to 3 0mL, 10-25mL, 10-20mL, 10-15mL, 15-40mL, 15-35mL, 15-30mL, 15-25mL, 15-20mL, 20-40mL, 20-35mL, 20-30mL, 20-25mL, 25-40mL, 25-35mL, 25-30mL, 30-40mL, 30-35mL or 35-40mL, or about 1-3 The present invention is configured to surround at least a portion of a chromatography column capable of accommodating a bed volume of 5 mL, 1 to 30 mL, 1 to 25 mL, 1 to 20 mL, 1 to 15 mL, 1 to 10 mL, 1 to 5 mL, 5 to 40 mL, 5 to 35 mL, 5 to 30 mL, 5 to 25 mL, 5 to 20 mL, 5 to 15 mL, 5 to 10 mL, 10 to 40 mL, 10 to 35 mL, 10 to 30 mL, 10 to 25 mL, 10 to 20 mL, 10 to 15 mL, 15 to 40 mL, 15 to 35 mL, 15 to 30 mL, 15 to 25 mL, 15 to 20 mL, 20 to 40 mL, 20 to 35 mL, 20 to 30 mL, 20 to 25 mL, 25 to 40 mL, 25 to 35 mL, 25 to 30 mL, 30 to 40 mL, 30 to 35 mL, or 35 to 40 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column capable of accommodating a bed volume of 15-25 mL or about 15-25 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column capable of accommodating a bed volume of 15-20 mL or about 15-20 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column capable of accommodating a bed volume of 18-20 mL or about 18-20 mL.

[0280] In some embodiments, at least a portion of the jacket member is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, the jacket member is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, the jacket member is configured to contact, e.g., be in direct contact with, the chromatography column.

[0281] In some embodiments, at least a portion of the jacket member is configured to be out of contact with at least a portion of the chromatography column. In some embodiments, at least a portion of the jacket member is configured to be out of contact with the chromatography column. In some embodiments, the jacket member is configured to be out of contact with the chromatography column.

[0282] In some embodiments, the jacket member is configured to allow an insulating layer to be disposed between one or more jacket components and the chromatography column. In some embodiments, the jacket member further comprises an insulating layer. In some embodiments, the insulating layer is configured to be disposed between one or more jacket components and at least a portion of the chromatography column. In some embodiments, the insulating layer is configured to be disposed between one or more jacket components and the chromatography column. In some embodiments, the insulating layer is configured to surround at least a portion of the chromatography column.

[0283] In some embodiments, the insulating layer comprises a gas layer, e.g., an air layer. In some embodiments, the insulating layer comprises a liquid layer. In some embodiments, the insulating layer comprises a solid layer.

[0284] In some embodiments, the temperature control member can be configured to heat the stationary phase contained in the chromatography column to a target temperature of about 30° C. to about 39° C. (e.g., 37° C. or about 37° C.). In some embodiments, the starting temperature is about 2° C., about 4° C., about 8° C., about 12° C., about 16° C., about 20° C., about 24° C., about 28° C., about 32° C., about 36° C., or greater than about 36° C. In some embodiments, the temperature control member can be configured to heat the stationary phase to 37° C. or about 37° C. In some embodiments, the temperature control member can be configured to heat the stationary phase to about 2° C. or higher, about 4° C. or higher, about 8° C. or higher, about 12° C. or higher, about 16° C. or higher, about 20° C. or higher, about 24° C. or higher, about 28° C. or higher, about 32° C. or higher, about 36° C. or higher, about 37° C. or higher, about 38° C. or higher, about 39° C. or higher, or about 40° C. or higher. In some embodiments, the temperature control member can be configured to maintain the stationary phase at a target temperature. In some embodiments, the temperature control member can be configured to maintain the stationary phase at a target temperature ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C. In some embodiments, the temperature control member can be configured to maintain the stationary phase at 37°C ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C.

[0285] In some embodiments, the temperature control element can be configured to adjust or maintain the temperature of a stationary phase contained in a chromatography column. In some aspects, the temperature control element is configured to heat, e.g., uniformly heat, the stationary phase from a starting temperature (e.g., room temperature) to a target temperature of about 30° C. to about 39° C. (e.g., 37° C. or about 37° C.). In some aspects, the temperature control element is further configured to maintain the stationary phase at the target temperature.

[0286] In some embodiments, the jacket member can include a temperature sensor configured to measure the temperature of the stationary phase in the internal cavity. In one aspect, the temperature sensor is configured to couple to a monitoring / display unit. In some embodiments, the temperature sensor is configured to electrically connect to a power source. In some embodiments, the power source is external to the jacket member. In some embodiments, the jacket member further includes a power source.

[0287] In any of the above aspects, the temperature control member can include a heat source. Alternatively, in any of the above aspects, the temperature control member can be configured to operatively connect to a heat source external to the housing assembly.

[0288] In some embodiments, the temperature control member comprises a heating element. In some embodiments, the heating element is configured to heat the stationary phase evenly.

[0289] In any of the above embodiments, the temperature control member can include a heating element selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In one aspect, the heating element is an electric heating element. In some embodiments, the electric heating element includes a metal plate, a metal rod, a metal wire, or a combination thereof. In one aspect, the heating element is an electromagnetic induction heating element. In some embodiments, the electromagnetic induction heating element includes an induction heating coil surrounding a magnetizable core configured to provide heat to a stationary phase in the internal cavity. In one aspect, the heating element is a non-electric heating element.

[0290] In some embodiments, the non-electrical heating element comprises a heating flow path including an inlet and an outlet for a heated fluid, e.g., a heated liquid or gas. In some embodiments, the heating flow path is a heating coil. In some embodiments, the heated fluid is heated water. In some embodiments, the heated water inlet is configured to connect to an external reservoir of heated water.

[0291] In some embodiments, the heating element is an electric heating element. In some embodiments, the electric heating element is configured to be electrically connected to a power source. In some embodiments, the power source is external to the jacket member. In some embodiments, the jacket member further comprises a power source.

[0292] In some embodiments, the electric heating element comprises a metal plate. In some embodiments, the metal plate is made at least in part of a thermally conductive metal, such as aluminum or copper. In some embodiments, the metal plate is made at least in part of aluminum (e.g., entirely of aluminum). In some embodiments, the electric heating element further comprises an electrical isolation layer, e.g., between at least a portion of the metal plate and at least a portion of another component of the electric heating element. In some embodiments, the electrical isolation layer lines at least a portion of one side of the metal plate (e.g., lines the entire one side of the metal plate).

[0293] In some embodiments, at least a portion of the heating element is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, the heating element is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, the heating element is configured to contact, e.g., be in direct contact with, the chromatography column.

[0294] In some embodiments, at least a portion of the heating element is configured to be out of contact with at least a portion of the chromatography column. In some embodiments, the heating element is configured to be out of contact with at least a portion of the chromatography column. In some embodiments, the heating element is configured to be out of contact with the chromatography column.

[0295] In some embodiments, the heating element is configured to surround at least a portion of the chromatography column.

[0296] In some embodiments, the temperature control member comprises multiple heating elements. In some embodiments, the temperature control member comprises 2 to 10 or about 2 to 10 heating elements, 2 to 8 or about 2 to 8 heating elements, 2 to 6 or about 2 to 6 heating elements, or 2 to 4 or about 2 to 4 heating elements, inclusive. In some embodiments, the temperature control member comprises two heating elements. In some embodiments, the temperature control member comprises three heating elements.

[0297] In some embodiments, the multiple heating elements are configured to heat the stationary phase evenly.

[0298] In some embodiments, each of the plurality of heating elements is selected from the group consisting of an electric heating element, an electromagnetic induction heating element, a non-electric heating element, and any combination thereof. In some embodiments, the plurality of heating elements are identical. In some embodiments, the plurality of heating elements is a combination of different heating elements.

[0299] In some embodiments, the plurality of heating elements comprises a plurality of non-electric heating elements. In some embodiments, the plurality of heating elements comprises a plurality of heating flow paths. In some embodiments, each of the plurality of heating flow paths has an inlet and an outlet for a heated fluid, for example, heated water. In some embodiments, at least two of the plurality of heating flow paths are fluidly connected to each other. In some embodiments, the plurality of heating flow paths are fluidly connected to each other. In some embodiments, the inlet of at least one of the plurality of heating flow paths is configured to connect to an external reservoir of heated liquid. In some embodiments, the inlet of each of the plurality of heating flow paths is configured to connect to an external reservoir of heated liquid.

[0300] In some embodiments, the plurality of heating elements comprises a plurality of electric heating elements, e.g., an electric heating element comprising a metal plate. In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to each other. In some embodiments, the plurality of electric heating elements are electrically coupled to each other. In some embodiments, at least one of the plurality of electric heating elements is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly. In some embodiments, each of the plurality of electric heating elements is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0301] In some embodiments, at least a portion of at least one of the plurality of heating elements is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, at least one of the plurality of heating elements is configured to contact, e.g., be in direct contact with, at least a portion of the chromatography column. In some embodiments, at least one of the plurality of heating elements is configured to contact, e.g., be in direct contact with, the chromatography column. In some embodiments, the plurality of heating elements is configured to contact, e.g., be in direct contact with, the chromatography column.

[0302] In some embodiments, at least a portion of at least one of the plurality of heating elements is configured to be out of contact with at least a portion of the chromatography column. In some embodiments, at least a portion of at least one of the plurality of heating elements is configured to be out of contact with the chromatography column. In some embodiments, at least one of the plurality of heating elements is configured to be out of contact with the chromatography column. In some embodiments, the plurality of heating elements is configured to be out of contact with the chromatography column.

[0303] In some embodiments, the plurality of heating elements are configured to be evenly or approximately evenly distributed around the chromatography column, for example, around the circumference of the chromatography column.

[0304] In some embodiments, the temperature control member comprises a non-electric heating element, e.g., a heating flow path for a heated fluid. In some embodiments, the temperature control member comprises multiple non-electric heating elements. In some embodiments, the jacket member comprises at least one opening per inlet and at least one opening per outlet for the heated fluid, e.g., for heated water. In some embodiments, the jacket member comprises at least two openings for inlets and / or at least two openings for outlets for the heated fluid, e.g., for heated water. In some embodiments, the jacket member is configured to electrically connect to a power source, e.g., a power source external to the housing assembly or a power source included in the housing assembly.

[0305] In some embodiments, the jacket member comprises an electric heating element, e.g., an electric heating element comprising a metal plate. In some embodiments, the jacket member comprises a plurality of electric heating elements. In some embodiments, the electric heating elements are configured to electrically connect to a power source. In some embodiments, at least one of the plurality of electric heating elements is configured to electrically connect to a power source. In some embodiments, each of the plurality of electric heating elements is configured to electrically connect to a power source.

[0306] In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to each other. In some embodiments, the plurality of electric heating elements are electrically coupled to each other.

[0307] In some embodiments, the jacket member includes two or more jacket parts, e.g., 2 to 10 or about 2 to 10 jacket parts, 2 to 8 or about 2 to 8 jacket parts, 2 to 6 or about 2 to 6 jacket parts, or 2 to 4 or about 2 to 4 jacket parts, inclusive. In some embodiments, the jacket member includes two jacket parts. In some embodiments, the jacket member includes three jacket parts. In some embodiments, the jacket member includes four jacket parts.

[0308] In some embodiments, at least two of the two or more jacket components each comprise a heating element. In some embodiments, the two or more jacket components each comprise a heating element.

[0309] In some embodiments, at least two of the two or more jacket components each comprise a temperature sensor. In some embodiments, the two or more jacket components each comprise a temperature sensor.

[0310] In some embodiments, at least two of the two or more jacket components each include a non-electric heating element. In some embodiments, at least two of the two or more jacket components each include a heated flow channel with an inlet and an outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include a heated flow channel with an inlet and an outlet for a heated fluid, e.g., heated water.

[0311] In some embodiments, at least two heating channels of the two or more jacket components are fluidly connected to one another. In some embodiments, the heating channels of the two or more jacket components are fluidly connected to one another.

[0312] In some embodiments, at least one of the two or more jacket components includes an opening for an inlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for an inlet for a fluid, e.g., heated water.

[0313] In some embodiments, at least one of the two or more jacket components includes an opening for an outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for an outlet for a fluid, e.g., heated water.

[0314] In some aspects, provided herein is a jacket member for column chromatography, comprising one or more jacket components configured to be removably connected to and surround at least a portion of a chromatography column, where the chromatography column is configured to house a stationary phase; and a temperature control member including one or more heating elements, where the one or more heating elements are configured to provide heat to the stationary phase, and where the temperature control member is configured to regulate or maintain a temperature of the stationary phase.

[0315] In some aspects, provided herein is a jacket member for column chromatography, comprising two or more jacket components configured to be removably connected and surround at least a portion of a chromatography column, wherein the chromatography column is configured to house a stationary phase; and a temperature control member including one or more heating elements, wherein the one or more heating elements are configured to provide heat to the stationary phase, and the temperature control member is configured to regulate or maintain a temperature of the stationary phase.

[0316] In some aspects, provided herein is a jacket member for column chromatography, comprising two jacket parts configured to be detachably connected and surround at least a portion of a chromatography column, wherein the chromatography column is configured to house a stationary phase; and a temperature control member including two heating elements that are heating coils, wherein one or more heating elements are configured to provide heat to the stationary phase, and wherein the temperature control member is configured to regulate or maintain a temperature of the stationary phase.

[0317] In some aspects, provided herein is a jacket member for column chromatography, comprising three jacket components configured to be removably connected and surround at least a portion of a chromatography column, the chromatography column configured to house a stationary phase; and a temperature control member including three heating elements that are electric heating elements, wherein one or more of the heating elements are configured to provide heat to the stationary phase, and the temperature control member is configured to regulate or maintain the temperature of the stationary phase.

[0318] II. Methods for Selecting, Stimulating, and / or Modifying Cells Using the devices disclosed herein, methods are provided herein for generating an output population (also referred to as an output composition) of cells, e.g., selected and stimulated CD3+ T cells, CD4+ T cells, and / or CD8+ T cells, comprising steps for cell selection, stimulation, and collection. In certain embodiments, the methods provided herein are used in connection with the manufacture, production, or production of cellular therapeutics. In some embodiments, the method for generating or producing an output composition, e.g., a method for generating or producing selected and stimulated T cells, comprises one or more of the following steps: isolating cells from a subject, incubating the cells under stimulating conditions, and genetically modifying the cells. In some embodiments, the method comprises sequential processing steps in which input cells, e.g., primary CD4+ and CD8+ T cells, are isolated, e.g., selected or separated, from a biological sample in a single step, incubated under stimulating conditions, and collected; then genetically modified to introduce a recombinant polynucleotide encoding a recombinant receptor into the cells, e.g., by transduction or transfection; and then collected, harvested, or packaged as the output population into a container, e.g., a bag or vial. In some embodiments, the cells of the output population are reintroduced into the same subject, optionally after cryopreserving and storing the cells. In some embodiments, the output population of modified cells is suitable for use in therapy, e.g., autologous cell therapy.

[0319] Using the devices disclosed herein, methods are provided herein for selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating the immobilized cells on the stationary phase (also referred to herein as on-column stimulation), and collecting and / or eluting the selected and stimulated cells that spontaneously detach from the stationary phase without using a competitor or free binder to facilitate detachment. Among the provided methods are methods that involve selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating the immobilized cells on the stationary phase, and collecting and / or eluting the selected and stimulated cells by gravity flow. In provided embodiments, stimulating target cells (e.g., CD3+, CD4+, or CD8+ T cells) on the stationary phase of a chromatography column promotes downregulation of the molecule used for cell selection (i.e., the selection marker), resulting in spontaneous detachment or release of the cells from the stationary phase. Cell detachment or release can occur without any additional steps or reagents. In some aspects, cells can be collected by gravity flow, such as by adding media or other solutions to the chromatography column. In particular embodiments, the added media or other solutions do not contain competitors or free binders to promote detachment of the cells from the stationary phase.

[0320] In particular embodiments, the provided methods are carried out to select and stimulate T cells. In some embodiments, T cells are selected from a biological sample, e.g., an apheresis sample, by applying the cells of the sample to an affinity chromatography matrix (e.g., a stationary phase) to which a selection agent specific for T cells or a subset thereof is immobilized or bound, e.g., as described in Section II.B-1. In provided embodiments, the method includes stimulating the cells immobilized on the stationary phase in the presence of one or more T cell stimulators. In some embodiments, the one or more stimulators include an agent for delivering a stimulatory signal in the T cells. In some embodiments, the stimulatory signal is a signal by a TCR / CD3 complex in the T cells, a CD3-containing complex in the T cells, and / or an ITAM-containing molecule in the T cells. In some embodiments, the stimulator (e.g., a first stimulator) is an agent that binds to CD3, such as an anti-CD3 antibody. In some embodiments, the one or more stimulators further include a second stimulator that further stimulates or enhances a signal in the T cells. In some embodiments, the second stimulatory agent is capable of specifically binding to one or more costimulatory molecules on T cells, such as CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the second stimulatory agent is an agent that binds to CD28, such as an anti-CD28 antibody. In some embodiments, the one or more stimulatory agents comprise an anti-CD3 antibody and an anti-CD28 antibody, e.g., an anti-CD3 Fab and an anti-CD28 Fab. In some embodiments, the one or more stimulatory agents are immobilized or bound to a reagent (e.g., a stimulatory reagent) that is added to the chromatography column. In particular embodiments, the stimulatory reagent is a soluble polymer or oligomeric reagent. For example, the one or more stimulatory agents are functionalized to an oligomeric or polymeric protein as opposed to a solid surface (e.g., a bead). Exemplary oligomeric stimulation reagents for use in the provided methods are described herein, eg, in Section II.B-2.In some embodiments, the oligomeric stimulatory reagent is an oligomeric streptavidin mutein functionalized or multimerized with one or more stimulatory agents (e.g., anti-CD3 Fab and anti-CD28 Fab). In the provided methods, the selected and stimulated T cells are collected by eluting or washing the selected and stimulated cells by gravity flow.

[0321] In some embodiments, the collecting step comprises washing the stationary phase with medium (e.g., serum-free medium) that does not contain a competitor or free binder for eluting the target cells (e.g., T cells) from the stationary phase. In some embodiments, the collecting step by gravity flow comprises adding medium to the stationary phase that does not contain a competitor or free binder for eluting the T cells from the stationary phase. In some embodiments, the composition containing the stimulated T cells does not contain a competitor or free binder. In some embodiments, the competitor or free binder is or contains biotin or a biotin analog, e.g., D-biotin. In some embodiments, the competitor or free binder is D-biotin. In some embodiments, the medium for washing the column to elute the cells by gravity flow is serum-free medium that contains a recombinant cytokine (e.g., IL-2).

[0322] In some embodiments, the method further comprises introducing a recombinant nucleic acid molecule encoding a recombinant protein into the stimulated T cells of the composition, thereby producing a composition comprising the transduced T cells. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a chimeric antigen receptor.

[0323] In some embodiments, the method includes further incubating the composition containing the stimulated cells (e.g., stimulated T cells). In some embodiments, the method includes further incubating the composition containing cells introduced with a recombinant receptor (e.g., transduced T cells). In some embodiments, the further incubation is carried out at 37°C ± 2°C or about 37°C ± 2°C. In some embodiments, the further incubation is carried out under conditions that do not allow or do not substantially allow cell expansion. In some embodiments, the further incubation is carried out under conditions for cell expansion (e.g., proliferation). In some embodiments, the further incubation is carried out in the presence of an additional agent capable of delivering a signal to the T cells. In some embodiments, the additional agent is contained in the medium used to wash the stationary phase. In some embodiments, the additional agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, the additional agent is a cytokine selected from among IL-2, IL-15, and IL-7. In some embodiments, the further incubation is carried out for a period of 72 hours, 48 ​​hours or less, 24 hours or less, or 12 hours or less.

[0324] In particular embodiments, methods are provided herein relating to generating an output population of cells expressing a recombinant receptor from an initial or input population of cells using the devices disclosed herein. In some embodiments, the input population of cells is produced, generated, and / or created by mixing, blending, and / or pooling cells, including from a population of cells containing enriched T cells, enriched CD4+ T cells, and / or enriched CD8+ T cells (hereinafter also referred to as an enriched population of T cells, an enriched population of CD4+ T cells, and an enriched population of CD8+ T cells, respectively). In some embodiments, the input population of cells is a mixed, blended, and / or pooled population of CD4+ T cells and CD8+ T cells. In certain embodiments, the provided methods are used in connection with genetically modifying selected and stimulated cells, for example, by introducing a polynucleotide encoding a recombinant protein by transduction or transfection. In certain embodiments, the methods can be used to isolate and select cells from a biological sample (e.g., whole blood, apheresis), such as a biological sample collected, harvested, and / or obtained from a subject, for the purpose of generating an input population of enriched T cells. In some embodiments, the provided methods can be used in conjunction with harvesting, collecting, and / or formulating a population of enriched T cells after modifying, transducing, and / or culturing the cells.

[0325] In certain embodiments, methods are provided herein relating to the introduction, e.g., introduction or transfection, of heterologous or recombinant polynucleotides into cells, such as by the methods described herein, for example, in Sections II-F, using the devices disclosed herein. In certain embodiments, the cells are incubated during or after genetic modification of the cells, for example, for a period sufficient to allow the incorporation of heterologous or recombinant polynucleotides encoding recombinant proteins or to allow the expression of recombinant proteins. In certain embodiments, the cells are incubated for a fixed or defined period of time, for example, for more than 18 hours or less than 4 days. In some embodiments, the modification process is initiated or started within a certain period of time from the time the stimulation process is initiated or started, for example, within 24 hours from the time the cells are exposed to the stimulating substance.

[0326] In some embodiments, the one or more processes are carried out, at least in part, in serum-free medium. In some embodiments, the serum-free medium is a defined or well-defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors. In some embodiments, the serum-free medium comprises cytokines. In some embodiments, the serum-free medium comprises cytokines or recombinant cytokines. In some embodiments, the serum-free medium comprises recombinant IL-2, IL-15, and / or IL-7. In some embodiments, the serum-free medium comprises glutamine. In some embodiments, the serum-free medium comprises glutamine and recombinant IL-2, IL-15, and IL-7.

[0327] In some embodiments, using the devices disclosed herein, methods are provided herein in which one, more, or all steps in preparing cells for clinical use, such as in adoptive cell therapy, are performed without exposing the cells to non-sterile conditions. In some embodiments, cell selection, stimulation, transduction, washing, and formulation are all performed within a closed, sterile system or device. In some embodiments, one or more of the steps are performed outside of a closed system or closed device. In some such embodiments, cells are transferred from a closed system or closed device under sterile conditions to another closed system, e.g., by aseptic transfer.

[0328] In some embodiments, the methods provided herein are carried out using any of the devices described in Section I.

[0329] In particular embodiments, the sample and / or an isolated portion of the sample (e.g., buffy coat, population of enriched T cells) can be collected, formulated for cryoprotection, frozen (e.g., cryoprotected), and / or stored below 0° C., below −20° C., or −70° C. or −80° C., or below −70° C. or −80° C. before, during, or after any stage or step of the methods provided herein. In some embodiments, the cells can be stored for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or for less than 1, 2, 3, 4, 5, 6, 7, 8 weeks, or for a period of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for more than 8 weeks. After storage, the sample or an isolated portion of the sample can be thawed and processing according to the method can be resumed at the same point in the process. In certain embodiments, the cultured and / or formulated population of enriched T cells is cryoprotected and stored prior to administration to a subject, eg, as an autologous cell therapy.

[0330] In particular embodiments, a portion of cells can be sampled or collected at any stage or step in the process, for example, cells can be collected from a population of cells (e.g., a population of T cells) while the population remains in a closed system. In certain embodiments, such cells can be analyzed for markers, traits, or characteristics, including but not limited to, viability, apoptosis, activation, stimulation, growth, and / or exhaustion. In some embodiments, cells are sampled or collected by an automated process. In some embodiments, analysis of sampled or collected cells is automated. In particular embodiments, analysis is performed in a closed system under sterile conditions.

[0331] In some embodiments, cells or populations of cells produced and / or processed by the provided methods may be compared to cells or populations of cells processed or produced by exemplary and / or alternative processes. In certain embodiments, the alternative and / or exemplary processes may differ in one or more specific aspects but otherwise include similar or identical attributes, aspects, steps, stages, reagents, or conditions as the embodiment or aspect of the provided methods to which the exemplary or alternative processes are being compared. For example, selected and stimulated cells, e.g., cellular output compositions, produced by the provided methods may be compared to cells produced by a process involving separate selection and stimulation steps that require the use of a competitor or free binder to detach the selected cells from the stationary phase. In some embodiments, unless otherwise specified, the provided methods and exemplary or alternative processes will otherwise be similar and / or identical, e.g., will involve similar or identical steps of selection, enrichment, stimulation, manipulation, transfection, transduction, culture, and / or formulation. In some embodiments, unless otherwise specified, the provided methods and alternative processes select and / or enrich cells from the same or similar type of biological sample and / or process cells and / or input cells of the same cell type.

[0332] In some embodiments, the selected and stimulated cells are a composition containing stimulated T cells, in which T cells have been selected from a biological sample (e.g., an apheresis sample or a whole blood sample) containing a plurality of T cells. In some embodiments, collection and / or elution of selected and stimulated cells that spontaneously detach from the stationary phase is accomplished by gravity flow, e.g., during a wash step. In the methods provided herein, cell selection, stimulation, and collection and / or elution steps are integrated, and no separate steps are required to facilitate detachment of the selected and stimulated cells from the stationary phase or to remove agents used to facilitate detachment (e.g., competitors and / or free binders). Thus, the methods reduce the number of processing steps required to produce a composition of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, subsequent incubation, stimulation, and / or selection (e.g., initial selection and / or polishing)), thereby reducing production time, minimizing potential cell stress, and reducing the possibility of contamination.

[0333] In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within 24 hours. In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours. In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 6, 5, 4, 3, or 2 hours. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 6 hours or less than about 6 hours. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 5.5 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 5 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 4.5 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 4 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 3 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 3-6 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., about 4-6 hours or less.In some embodiments, the methods produce an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 5-6 hours or less than about 5-6 hours. In some embodiments, the methods produce an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 4-5 hours or less than about 4-5 hours. In some embodiments, the methods provided herein produce a composition of engineered T cells (e.g., a therapeutic cell composition) within 5 days. In some embodiments, the methods provided herein produce a composition of engineered T cells (e.g., a therapeutic cell composition) in 4-5 days or about 4-5 days. In some embodiments, the processes provided herein result in a manufacturing process that is 4 or 5 days in length. In some embodiments, the processes provided herein result in a manufacturing process that is about 4-5 days in length. In some embodiments, the processes provided herein result in a manufacturing process that is 4 days or about 4 days or 96±6 hours in length.

[0334] The provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., other cells in a sample, and immobilizing the cells on a stationary phase of a chromatography column; stimulating the selected cells immobilized on the stationary phase; and collecting the selected and stimulated cells in the absence of processing steps to detach the cells from the stationary phase and to remove from the output composition of the selected and stimulated cells any agents (e.g., competitors or free binders) used to promote detachment. In particular embodiments, the provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., other cells in a sample, and immobilizing the cells on a stationary phase of a chromatography column; stimulating the selected cells immobilized on the stationary phase; and eluting and / or collecting the selected and stimulated cells by gravity flow.

[0335] In certain aspects, the methods provided herein are an improvement over many existing methods for generating engineered cells (e.g., T cells), such as for cell therapy, that include one or more additional steps after cell selection (e.g., immunoaffinity-based selection) and prior to cell stimulation. In some embodiments, the one or more additional steps present in existing methods can include one or more elution steps using competitive or free-binding agents to recover or harvest the selected cells and / or steps to remove reagents used for selection (e.g., magnetic bead reagents or antibodies). In some embodiments, such additional steps may lengthen the process for genetically engineering cells for cell therapy and / or may result in manipulation of the cells during the process that can impact the differentiation state, viability, or cell number of the cells. In certain aspects, the provided methods produce a population of selected and stimulated cells in a reduced amount of time compared to methods that include separate selection and stimulation steps, requiring additional steps to detach the cells from the stationary phase and to remove agents used to promote detachment.

[0336] In certain aspects, the methods produce an output population (also referred to as an output composition) of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 24 hours of initiating on-column stimulation, also referred to herein as on-column stimulation. In some embodiments, the methods produce an output population (e.g., an output composition) of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within or about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours of initiating on-column stimulation. In some embodiments, the methods generate an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within or about 6, 5, 4, 3, or 2 hours. In some embodiments, the methods generate an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within or about 3-6 hours. In some embodiments, the methods generate an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within or about 4-6 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and / or subsequent incubation, stimulation and / or selection rounds (e.g., polishing)) within or about 5-6 hours.In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 4 to 5 hours or about 4 to 5 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 6 hours or about 6 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 5.5 hours or about 5.5 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 5 hours or about 5 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 4.5 hours or about 4.5 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 4 hours or about 4 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, and / or subsequent incubation, stimulation, and / or selection rounds (e.g., polishing)) within 3 hours or about 3 hours.

[0337] In some embodiments, the method involves the use of a stimulatory agent capable of delivering a stimulatory signal to cells by binding to a molecule on the surface of the cells. In some embodiments, the stimulatory agent is included in an oligomeric stimulatory reagent (e.g., a streptavidin mutein oligomer conjugated to an anti-CD3 Fab and an anti-CD28 Fab) that can be added to the stationary phase. In some embodiments, the stimulation results in the spontaneous detachment of selected cells from the stationary phase, thereby allowing for collection and / or elution of the selected, stimulated cells without additional processing steps to detach the cells from the stationary phase and to remove the agent used to promote detachment from the stimulated cell output composition. In some embodiments, the stimulation results in the spontaneous detachment or release of selected cells from the stationary phase, thereby allowing for collection and / or elution of selected, stimulated cells by gravity flow. In some embodiments, gravity flow relies on collecting or eluting cells that have spontaneously detached from a column (e.g., a stationary phase). In some embodiments, a wash step may be used, e.g., in combination with gravity flow, to elute spontaneously detached cells from the column (e.g., a stationary phase). In some embodiments, the washing step can simply involve adding cell culture medium (e.g., serum-free medium) to the column, e.g., the same medium that is present in the cell input composition before adding or immobilizing the cells to the stationary phase. In certain aspects, the method successfully produces an uncontaminated (e.g., free of agents used for detachment (e.g., competitors, free binders) and / or selection agents) composition of selected, stimulated cells suitable for further processing, e.g., genetic manipulation, expansion, incubation, or subsequent stimulation and / or selection rounds (e.g., polishing), within 24 hours of initiating on-column stimulation. Articles of manufacture and devices thereof are also provided.

[0338] A variety of methods are available for generating cell populations suitable for use in cell therapy (e.g., selected (enriched), stimulated cells engineered to express a recombinant protein (e.g., a chimeric antigen receptor)). However, in some aspects, these methods can require long or relatively long times to generate the cells, at least in part due to the need to perform multiple processing steps. Multiple processing steps can also result in cellular stress, thus affecting the usefulness of the cells for downstream processing. Additional methods for generating cell compositions are needed.

[0339] In certain aspects, provided methods are based on the discovery that selecting and stimulating target cells (e.g., CD3+, CD4+, or CD8+ T cells) on a stationary phase of a chromatography column promotes downregulation of the molecule (i.e., selection marker) used for cell selection, resulting in spontaneous detachment of the cells from the stationary phase. In some embodiments, the stationary phase of the chromatography column is functionalized with an agent (e.g., a selection agent) capable of specifically binding to a molecule (e.g., a selection marker) on the surface of the target cells. In this manner, when a sample containing target cells bearing a selection marker (e.g., CD3, CD4, CD8) is mixed with the stationary phase (e.g., adding the sample to the stationary phase), the target cells (e.g., CD3+, CD4+, CD8+ T cells) are indirectly immobilized to the stationary phase. In certain aspects, the target cells (e.g., T cells) are stimulated while immobilized on the stationary phase (e.g., on-column stimulation), for example, by adding a stimulatory agent, a stimulatory reagent containing the stimulatory agent, and / or via a stimulatory agent directly or indirectly coupled to the stationary phase. In particular embodiments, the stimulatory agent comprises an agent that activates or stimulates T cells, e.g., an anti-CD3 / anti-CD28 antibody (e.g., Fab) agent. Thus, in some aspects, the provided methods and other embodiments are advantageous in that they simplify multiple processing steps (e.g., selection and stimulation) and / or eliminate processing steps (e.g., steps to remove selection reagents and / or selection agents used to promote detachment), allowing the simplified process to occur in the same container and / or the same closed system, which can result in improved efficiency and sterility.

[0340] In certain aspects, the method involves the use of an oligomeric stimulatory reagent comprising a stimulatory substance capable of delivering a stimulatory signal to target cells (e.g., T cells). Exemplary oligomeric reagents include streptavidin mutein oligomers reversibly bound or conjugated to one or more antibodies or fragments thereof capable of delivering a stimulatory signal to target cells, e.g., T cells. In some embodiments, the oligomeric stimulatory reagent is a streptavidin mutein oligomer conjugated to an anti-CD3 Fab and an anti-CD28 Fab. Existing reagents used to stimulate T cells in vitro, for example, in the absence of exogenous growth factors or in the presence of small amounts of exogenous growth factors, are known (see, for example, U.S. Patent No. 6,352,694 (B1) and European Patent EP0700430B1). Generally, such reagents use beads with a diameter of more than 1 μm, e.g., magnetic beads, to which various binding agents (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies) are immobilized. However, such magnetic beads can be difficult to incorporate into methods for stimulating cells, for example, under conditions required for clinical trials or therapeutic purposes, because it is necessary to ensure that these magnetic beads are substantially or completely removed before administering engineered T cells to a subject. In some aspects, such removal, for example, by exposing the cells to a magnetic field, can reduce the yield of viable cells available for cell therapy. In certain instances, such reagents, e.g., stimulation reagents containing magnetic beads, must be incubated with cells for a minimum time to allow for the detachment of a sufficient number of T cells from the stimulation reagent. Furthermore, reagents such as beads are not easily compatible with column chromatography due to their physical properties.

[0341] The provided methods utilize oligomeric stimulating reagents (e.g., streptavidin mutein oligomers conjugated to anti-CD3 and anti-CD28 antibodies, e.g., Fabs) to overcome such potential limitations. For example, in some embodiments, the provided methods include adding a soluble oligomeric reagent that is not bound to a solid support (e.g., beads) to the stationary phase to initiate stimulation. In some embodiments, the provided methods can include steps to reduce or minimize the amount of residual oligomeric stimulating reagent that may be present at the end of the overall process of engineering cells for cell therapy. In some embodiments, the use of oligomeric reagents reduces or avoids the risk of residual reagent in output cells, e.g., engineered cells, produced or generated by the methods. This is because the addition of a competing reagent or free binding agent can be used to dissociate (e.g., disrupt binding to) the oligomeric stimulating reagent from the stimulatory agent in the cell-containing composition. In some embodiments, because the oligomeric stimulating reagent is soluble, for example, adding a competing reagent or free binding agent is not necessary; it may be sufficient to simply reduce or remove the oligomeric stimulating reagent from the cells in the composition by one or more wash steps. In some embodiments, this also means that a process that complies with GMP standards can be more ea...

Claims

1. 1. A chromatography column for column chromatography, comprising a housing assembly, the housing assembly comprising: an inlet housing member and an outlet housing member, at least the inlet housing member and the outlet housing member defining an interior cavity configured to accommodate a stationary phase for column chromatography, wherein: the internal cavity contains a stationary phase for chromatography; and the stationary phase comprises an affinity chromatography matrix; an inlet housing member and an outlet housing member; a temperature control member configured to provide heat to the stationary phase in the internal cavity; a connector configured to operatively connect the internal cavity to a gas source, thereby enabling or achieving the introduction of gas into the internal cavity; A chromatography column comprising:

2. 10. The chromatography column of claim 1, wherein the housing assembly further comprises a sidewall member, the inlet housing member, the outlet housing member and the sidewall member forming an interior cavity.

3. the inlet housing member includes one or more inlets operatively connected to the internal cavity to allow for the intake of an input composition into the internal cavity; and / or the outlet housing member includes one or more outlets operatively connected to the internal cavity to enable or effect discharge of the output composition from the internal cavity; 3. The chromatography column according to claim 1 or 2.

4. 4. The chromatography column of claim 1, wherein the temperature control member is configured to heat the stationary phase to a target temperature of between 30°C and 39°C.

5. 4. The chromatography column of claim 1, wherein the temperature control member is configured to heat the stationary phase to a target temperature of between 35°C and 39°C.

6. 6. The chromatography column of claim 1, wherein the temperature control member comprises a heating element or a plurality of heating elements.

7. 7. The chromatography column of claim 6, wherein the heating element and / or at least one of the plurality of heating elements is an electric heating element.

8. 8. The chromatography column of claim 7, wherein the heating element and / or at least one of the plurality of heating elements is a metal plate, a metal rod, a metal wire, or a combination thereof.

9. 9. The chromatography column of any one of claims 6 to 8, wherein the heating element and / or at least one of the plurality of heating elements is an electromagnetic induction heating element comprising an induction heating coil surrounding a magnetizable core configured to impart heat to the stationary phase in the internal cavity.

10. 7. The chromatography column of claim 6, wherein the heating element and / or at least one of the plurality of heating elements is a non-electrical heating element comprising a heated flow path.

11. 11. The chromatography column of claim 10, wherein the heating element and / or at least one of the plurality of heating elements is a heating coil including an inlet and an outlet for a heated fluid.

12. 12. The chromatography column of claim 11, wherein the heated fluid is heated water.

13. 13. The chromatography column according to any one of claims 6 to 12, wherein the heating element and / or at least one of the heating elements is arranged outside the internal cavity.

14. 14. The chromatography column of claim 6, wherein the heating element and / or at least one of the plurality of heating elements surrounds at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the sidewall member.

15. 15. The chromatography column of any one of claims 6 to 14, wherein the plurality of heating elements are evenly distributed around the circumference of the sidewall member.

16. 16. The chromatography column of any one of claims 6 to 15, wherein the housing assembly further comprises a jacket member comprising a heating element or at least one of a plurality of heating elements, the jacket member being configured to surround at least a portion of the inlet housing member, at least a portion of the outlet housing member and / or at least a portion of the sidewall member.

17. 17. The chromatography column of claim 16, wherein the jacket members are removably connected together and surround at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the sidewall member.

18. 18. The chromatography column of claim 16 or 17, wherein the jacket member is configured to completely surround the sidewall member.

19. 19. The chromatography column of any one of claims 16 to 18, wherein the jacket member comprises a plurality of heating elements, and at least two of the two or more jacket members each comprise at least one of the plurality of heating elements.

20. 20. The chromatography column of claim 19, wherein at least two of the two or more jacket members each further comprise a temperature sensor.

21. A chromatography column set comprising a plurality of chromatography columns according to any one of claims 1 to 20.

22. 22. The chromatography column or set of chromatography columns according to any one of claims 1 to 21, wherein the stationary phase comprises a non-magnetic, non-ferromagnetic or non-magnetic material.

23. 23. The chromatography column or set of chromatography columns according to any one of claims 1 to 22, wherein the stationary phase comprises a selected substance immobilized thereon.

24. 24. The chromatography column or set of chromatography columns of claim 23, wherein the selection agent has the ability to specifically bind to a selection marker on the surface of one or more cells.

25. 25. The chromatography column or set of chromatography columns of claim 24, wherein the one or more cells are immune cells or T cells.

26. 26. The chromatography column or set of chromatography columns of claim 24 or 25, wherein the selection substance is or comprises an antibody, an antibody fragment, or a proteinaceous binding molecule that specifically binds to the selection marker.

27. 27. The chromatography column or set of chromatography columns of any one of claims 23 to 26, wherein the selection substance comprises or is an anti-CD3 Fab, an anti-CD8 Fab, an anti-CD4 Fab or an anti-CD27 Fab.

28. 28. The chromatography column or set of chromatography columns of any one of claims 23 to 27, wherein the selection substance is indirectly bound to the stationary phase via a selection reagent to which the selection substance reversibly binds.

29. 29. The chromatography column or set of chromatography columns of claim 28, wherein the selection reagent comprises or is a mutein of streptavidin that reversibly binds to the streptavidin-binding peptide.

30. 30. The chromatography column or set of chromatography columns of any one of claims 23 to 29, further comprising one or more stimuli indirectly immobilized on the stationary phase via muteins of streptavidin that reversibly bind to the streptavidin-binding peptides.

31. 31. The chromatography column or set of chromatography columns of claim 30, comprising a first stimulatory substance and a second stimulatory substance, wherein the first stimulatory substance is an anti-CD3 Fab and the second stimulatory substance is an anti-CD28 Fab.

32. 32. The chromatography column or set of chromatography columns of claim 31, wherein the first stimulus substance and the second stimulus substance independently further comprise a streptavidin-binding peptide.

33. The streptavidin mutein contains the amino acid sequence Val at sequence positions corresponding to positions 44-47 of SEQ ID NO:

1. 44 -Thr 45 -Ala 46 -Arg 47 or the streptavidin mutein comprises the amino acid sequence Ile at a sequence position corresponding to positions 44-47 of SEQ ID NO:

1. 44 -Gly 45 -Ala 46 -Arg 47 33. The chromatography column or set of chromatography columns according to any one of claims 30 to 32, comprising:

34. 34. The chromatography column or set of chromatography columns according to any one of claims 30 to 33, wherein the streptavidin mutein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 27, 28, 104 and 105.

35. 35. A device comprising: (i) a chromatography column or set of chromatography columns according to any one of claims 1 to 34; and (ii) an input composition reservoir operatively connected to the interior cavity via an inlet in the inlet housing member.

36. A method for on-column stimulation of T cells, comprising the steps of: Incubating a sample comprising a plurality of T cells with one or more stimulatory substances to deliver a stimulatory signal in one or more T cells of the plurality of T cells immobilized on a stationary phase in the chromatography column or set of chromatography columns of any one of claims 1 to 34 or the device of claim 35; After the start of incubation, harvesting the one or more T cells from the stationary phase, thereby producing an output composition comprising stimulated T cells.

37. 37. The method of claim 36, wherein the selection agent of the stationary phase specifically binds to a selection marker on the surface of the plurality of T cells, and the immobilization of the plurality of T cells in the stationary phase is achieved by specific binding of the selection agent to the selection marker.

38. 38. The method of claim 36 or 37, wherein the temperature control member regulates the temperature of the stationary phase to a target temperature of between 30°C and 39°C during at least a portion of the incubation.

39. 39. The method of any one of claims 36-38, wherein the connector allows gas to be introduced into the internal cavity during at least a portion of the incubation.

40. 40. The method of claim 39, wherein the gas is sterile.

41. 41. The method of claim 39 or 40, wherein the gas is or comprises air.

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