CELL SELECTION AND / OR STIMULATION DEVICES AND METHODS OF USE

Description

Field

[0001] The present disclosure relates to cell selection and / or stimulation devices and methods of use. In some aspects, the selected and / or stimulated cells are useful for genetic engineering, and ultimately, cell therapy.Background

[0002] Various cell therapy methods are available for treating diseases and conditions. Among cell therapy methods are methods involving immune cells, such as T cells (e.g., CD4+ and CD8+ T cells), which may be genetically engineered with a recombinant receptor, such as a chimeric antigen receptors. US 2010 / 0005867 A1 discloses a temperature control unit for a fluidic device comprising a column which may be filled with a package composition. WO 2014 / 008058 A1 discloses a device for preforming liquid chromatography comprising a chromatography column and an insulating member surrounding the chromatography column. A plurality of heaters may heat the stationary phase of the column, which is placed in an internal cavity between two housing members. US 2017 / 0282096 A1 discloses also a device comprising a chromatographic column and a surrounding vacuum insulated jacket which could be used for heating. The chromatographic column may comprise two frits between the inlet / outlet and the stationary phase. Improved devices and methods for generating cell populations suitable for use, for example in cell therapy, are needed. Provided are device, articles of manufacture, and methods that meet such needs.Summary

[0003] In some embodiments, disclosed herein is 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 internal cavity configured to house 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 permitting or effecting intake 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 side wall member, and the inlet housing member, the outlet housing member, and the side wall member form the internal cavity.

[0004] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house 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 permitting or effecting intake of gas into the internal cavity.

[0005] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: a chromatography column comprising an internal cavity configured to house 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 permitting or effecting intake of gas into the internal cavity. In some embodiments, the chromatography column comprises an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form the internal cavity.

[0006] In any of the preceding embodiments, the connector can be disposed on the inlet housing member, the outlet housing member, and / or the side wall member.

[0007] In any of the preceding embodiments, the connector can be formed between any two or among all three of the inlet housing member, the outlet housing member, and the side wall member.

[0008] In any of the preceding embodiments, the housing assembly may comprise a plurality of the connectors.

[0009] In any of the preceding embodiments, the connector can be a bonded connector, a screw 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 preceding embodiments, the connector can be a luer lock connector or a luer slip connector. In any of the preceding embodiments, the connector can comprise a male fitting or a female fitting. In any of the preceding embodiments, the connector can be configured to sealingly engage tubing in fluid communication with the gas source. In any of the preceding embodiments, the connector can comprise one or more valve. In any of the preceding embodiments, the connector can be operably connected to tubing comprising one or more valve.

[0010] In any of the preceding embodiments, the connector can comprise one or more filter. In any of the preceding embodiments, the connector can be operably connected to tubing comprising one or more filter. In any of the preceding embodiments, the one or more filter can be a gas filter, e.g., an air filter. In any of the preceding embodiments, the one or more filter can be an air filter. In any of the preceding embodiments, the one or more filter can be a sterile filter and / or a sterilizing filter for sterilization by filtration. In any of the preceding embodiments, the one or more filter can be a sterile filter. In any of the preceding embodiments, the one or more filter can be a sterilizing filter for sterilization by filtration.

[0011] In any of the preceding embodiments, the inlet housing member can comprise an upper lid of the housing assembly. In some embodiments, the upper lid is removably attached to the inlet housing member or the side wall member. In some embodiments, the upper lid is integrally formed with the inlet housing member or the side wall member. In any of the preceding embodiments, the connector can be disposed on the upper lid.

[0012] In any of the preceding embodiments, the inlet housing member may comprise one or more inlet operably connected to the internal cavity to permit intake of an input composition into the internal cavity. In some embodiments, the one or more inlet is disposed on the upper lid. In some embodiments, the connector and the one or more inlet are disposed on the upper lid at the same or different locations.

[0013] In any of the preceding embodiments, fluid path through the one or more inlet can be at an angle of about 90 degrees to the upper lid, while fluid path through the connector can be at an angle of about 45 degrees to the upper lid.

[0014] In any of the preceding embodiments, the outlet housing member may comprise a lower lid of the housing assembly. In some embodiments, the lower lid is removably attached to the outlet housing member or the side wall member, or the lower lid is integrally formed with the outlet housing member or the side wall member.

[0015] In any of the preceding embodiments, the outlet housing member can comprise one or more outlet operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity. In some embodiments, the one or more outlet is disposed on the lower lid. In some embodiments, the connector and the one or more outlet are disposed on the lower lid at the same or different locations. In some embodiments, fluid path through the one or more outlet is at an angle of about 90 degrees to the lower lid.

[0016] In any of the preceding embodiments, the gas source can be or comprise a gas reservoir or an outside environment. In any of the preceding embodiments, gas in the gas source may be sterile. In any of the preceding embodiments, the gas can be or comprise air.

[0017] In any of the preceding embodiments, the housing assembly can further comprise tubing operably connected to the gas source. In some embodiments, the tubing is configured to sterilely connect the internal cavity to the gas source. In any of the preceding embodiments, the tubing can comprise one or more valve. In any of the preceding embodiments, the tubing may comprise one or more filter.

[0018] In any of the preceding embodiments, the housing assembly can further comprise one or more porous member, e.g., a cell strainer or a cell sieve. In some embodiments, the one or more porous member can be a cell strainer or a cell sieve. In some embodiments, the housing assembly comprises a first porous member configured to separate the stationary phase and an inlet of the internal cavity, and the first porous member is optionally between the inlet housing member and the side wall member. In some embodiments, the housing assembly further comprises a second porous member configured to separate the stationary phase and an outlet of the internal cavity, and the second porous member is optionally between the outlet housing member and the side wall member.

[0019] In any of the preceding embodiments, the housing assembly can comprise a first porous member configured to separate the stationary phase and an inlet of the internal cavity, wherein the first porous member is optionally between the inlet housing member and the side wall member; and / or a second porous member configured to separate the stationary phase and an outlet of the internal cavity, wherein the second porous member is optionally between the outlet housing member and the side wall member. In some embodiments, the first porous member or the second porous member is independently a cell strainer or a cell sieve.

[0020] In any of the preceding embodiments, the first porous member can be between the inlet housing member and the side wall member. In any of the preceding embodiments, the second porous member can be between the outlet housing member and the side wall member.

[0021] In any of the preceding embodiments, the one or more porous member may have an average pore diameter of about 20 µm, or the one or more porous member may comprise a mesh having a mesh size of about 20 µm.

[0022] In any of the preceding embodiments, the temperature control member may be configured to regulate or maintain a temperature of the stationary phase in the internal cavity. In any of the preceding 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 between about 35°C and about 39°C (e.g., at or at about 37°C). In any of the preceding embodiments, the target temperature can be at or about 37°C. In some embodiments, the temperature control member is further configured to maintain the stationary phase at the target temperature.

[0023] In any of the preceding embodiments, the temperature control member can be configured to heat the stationary phase to a target temperature between about 30°C and about 39°C. In any of the preceding embodiments, the target temperature can be between about 35°C and about 39°C, optionally at or about 37°C. In any of the preceding embodiments, the target temperature can be at or about 37°C. In some embodiments, the temperature control member is further configured to maintain the stationary phase at the target temperature.

[0024] In any of the preceding embodiments, the housing assembly can comprise a temperature sensor configured to measure the temperature of the stationary phase in the internal cavity. The temperature sensor may form a part of the temperature control member, or provided separately from the temperature control member. In some embodiments, the temperature sensor is configured to couple to a monitoring / display unit.

[0025] In any of the preceding embodiments, the temperature control member may comprise a heating source. In any of the preceding embodiments, the temperature control member may be configured to operably connect to a heating source which is external to the housing assembly.

[0026] In any of the preceding embodiments, the temperature control member can comprise a heating element or a plurality of heating elements.

[0027] In any of the preceding embodiments, the heating element and / or the plurality of heating elements can be configured to uniformly heat the stationary phase.

[0028] In any of the preceding 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 preceding 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 the 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-electric heating element comprises a heating channel comprising an inlet and an outlet for a heated fluid, e.g., a heated liquid or gas. In some embodiments, the heated fluid can be a heated liquid or a heated gas. In some embodiments, the heating channel can be a heating coil. In some embodiments, the heated fluid can be heated water. In some embodiments, the heating channel is a heating coil and the heated fluid is heated water. In some embodiments, the inlet for heated water is configured to connect to an external reservoir of heated water.

[0029] In any of the preceding embodiments, the heating element may be disposed along and / or around a central axis of the internal cavity. In any of the preceding embodiments, the heating element may 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, the heating element may be disposed inside the side wall member, outside the side wall member, or partially inside and partially outside the side wall member. In any of the preceding embodiments, the heating element can comprise a coil surrounding the inlet housing member, the outlet housing member, and / or the side wall member.

[0030] In any of the preceding embodiments, the heating element can comprise a heating channel surrounding the inlet housing member, the outlet housing member, and / or the side wall member. In any of the preceding embodiments, the heating element can comprise a heating coil surrounding the inlet housing member, the outlet housing member, and / or the side wall member. In any of the preceding embodiments, at least one of the plurality of heating elements may be disposed along and / or around a central axis of the internal cavity. In any of the preceding embodiments, at least one of the plurality of heating elements may 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 plurality of heating elements may be disposed inside the side wall member, outside the side wall member, or partially inside and partially outside the side wall member.

[0031] In any of the preceding embodiments, the heating element and / or at least one of the plurality of heating elements may 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. In any of the preceding embodiments, the heating element and / or at least one of the plurality of heating elements may surround at least a portion of the side wall member.

[0032] In any of the preceding embodiments, the plurality of heating elements can be uniformly or about uniformly distributed around the circumference of the side wall member.

[0033] 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 the side wall member, optionally at least a portion of the side wall 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 side wall member.

[0034] 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 not in contact with the inlet housing member, the outlet housing member, or the side wall member.

[0035] In any of the preceding embodiments, the housing assembly can further comprise an insulation layer between the heating element and / or the 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. In some embodiments, the insulation layer can comprise a gas, optionally air, or a liquid. In some embodiments, the insulation layer can comprise air.

[0036] In any of the preceding embodiments, the heating element can comprise 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 side wall member

[0037] In any of the preceding embodiments, the heating element can comprise 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 side wall member.

[0038] In any of the preceding embodiments, the plurality of heating elements can comprise a plurality of heating channels that surround at least a portion of inlet housing member, at least a portion of the outlet housing member, and / or at least a portion of the side wall member. In any of the preceding embodiments, at least two of the plurality of heating channels can be fluidly coupled to one another.

[0039] In any of the preceding embodiments, the plurality of heating elements can be a plurality of electric heating elements that 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. In any of the preceding embodiments, at least two of the plurality of electric heating elements can be electrically coupled to one another. In any of the preceding 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.

[0040] In any of the preceding embodiments, the housing assembly can further comprise a jacket member comprising the heating element or at least one of the plurality of heating elements, wherein 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 side wall member. In any of the preceding embodiments, the housing assembly can further comprise a jacket member comprising the temperature control member comprising the heating element or at least one of the plurality of heating elements, wherein 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 side wall member.

[0041] 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 side wall member.

[0042] In any of the preceding embodiments, the jacket member can be releasably 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 side wall member.

[0043] In any of the preceding embodiments, the jacket member can be configured to surround at least a portion of the side wall member, optionally can be configured to entirely surround the side wall member. In any of the preceding embodiments, the jacket member can be configured to entirely surround the side wall member. In any of the preceding embodiments, the jacket member may surround at least a portion of the side wall member, optionally may entirely surround the side wall member. In any of the preceding embodiments, the jacket member can be configured to entirely surround the side wall member. In any of the preceding embodiments, the jacket member may entirely surround the side wall member.

[0044] In any of the preceding embodiments, the jacket member can comprise two or more jacket components that are configured to together surround the at least a portion of the inlet housing member, the at least a portion of the outlet housing member, and / or the at least a portion of the side wall member, optionally entirely surround the side wall member. In any of the preceding embodiments, the jacket member can comprise two or more jacket components that together are configured to surround at least a portion of the side wall member. In any of the preceding embodiments, the jacket member can comprise two or more jacket components that together are configured to entirely surround the side wall member.

[0045] In any of the preceding embodiments, a portion of the one or more inlet of the inlet housing member and / or a portion of the one or more outlet of the outlet housing member can be exposed by the jacket member. In any of the preceding embodiments, a portion of the one or more inlet of the inlet housing member and / or a portion of the one or more outlet of the outlet housing member can be outside the jacket member.

[0046] 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 side wall member, optionally at least a portion of the side wall 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 side wall member.

[0047] In any of the preceding embodiments, at least a portion of the jacket member can be not in contact with the inlet housing member, the outlet housing member, or the side wall member.

[0048] In any of the preceding embodiments, the heating element or the at least one of the plurality of heating elements can be a heating channel comprising a inlet and an outlet for a heated fluid and the jacket member can comprise at least one opening for the inlet for the heating fluid and at least one opening for the outlet for the heated fluid.

[0049] In any of the preceding embodiments, the heating element or the 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.

[0050] In any of the preceding embodiments, the two or more jacket components can be configured to be releasably connected together.

[0051] In any of the preceding embodiments, the jacket member can comprise a plurality of heating elements and at least two of the two or more jacket components can each comprise at least one of the plurality of heating elements. In any of the preceding embodiments, the at least two of the two or more jacket components can each further comprise a temperature sensor

[0052] In any of the preceding embodiments, the at least two of the two or more jacket components can each comprise a heating channel comprising 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, the heating channels of the at least two 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 the heating channels of the at least two of the two or more jacket components can be configured to connect to an external reservoir of heated water.

[0053] In any of the preceding embodiments, the at least two of the two or more jacket components can each comprise an electric heating element, optionally an electric heating element that comprises a metal plate. In any of the preceding embodiments, the electric heating elements of the at least two of the two or more jacket components can be electrically coupled to one another. In any of the preceding embodiments, the electric heating elements of the at least two of the two or more jacket components can configured to electrically connect to a power source external to the housing assembly.

[0054] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house 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 permitting or effecting intake of gas into the internal cavity.

[0055] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising a heating element and 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 permitting or effecting intake of gas into the internal cavity.

[0056] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising 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 permitting or effecting intake of sterile gas into the internal cavity.

[0057] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising a heating element comprising a metal plate 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 permitting or effecting intake of sterile gas into the internal cavity.

[0058] In some embodiments, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising a heating element comprising 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 permitting or effecting intake of sterile gas into the internal cavity. In some embodiments, the heating coil comprises an inlet and an outlet for heated water.

[0059] In any of the preceding embodiments, the heating coil may surround the inlet housing member, the outlet housing member, and the side wall member.

[0060] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, where the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising 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 permitting or effecting intake of 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 preceding embodiments, the housing assembly may further comprise the gas filter.

[0061] In some embodiments, the chromatography column can comprise an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form the internal cavity. In some embodiments, the jacket member can be releasably 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 side wall member. In any of the preceding embodiments, the housing assembly can further comprise a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby permitting or effecting intake of sterile gas into the internal cavity.

[0062] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase, wherein the temperature control member comprises a heating coil configured to provide heat to the stationary phase; a jacket member comprising the heating coil, wherein the jacket member is releasably connected to surround at least a portion of the inlet housing member, the outlet housing member, and the side wall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby permitting or effecting intake of sterile gas into the internal cavity.

[0063] In some embodiments, the heating coil entirely surrounds the side wall member. In some embodiments, the jacket member can comprise a second heating coil, and the heating coil and the second heating coil can together surround the side wall member.

[0064] In some embodiments, provided herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase, wherein the temperature control member comprises an electric heating element that comprises a metal plate and is configured to provide heat to the stationary phase; a jacket member comprising the electric heating element , wherein the jacket member is releasably connected to surround at least a portion of the inlet housing member, the outlet housing member, and the side wall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby permitting or effecting intake of sterile gas into the internal cavity.

[0065] In some embodiments, the jacket member can comprise a plurality of electric heating elements that comprise metal plates and that are uniformly or about uniformly distributed around the circumference of the side wall member.

[0066] In some embodiments, disclosed herein is a housing assembly set, comprising a plurality of the housing assembly of any of the preceding embodiments. In some embodiments, the housing assembly set comprises at least two of the plurality of the housing assembly arranged sequentially. In any of the preceding embodiments, the housing assembly set may comprise at least two of the plurality of the housing assembly arranged in parallel.

[0067] 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.

[0068] In some embodiments, provided herein is a chromatography kit, comprising the housing assembly or the housing assembly set of any of the preceding embodiments, and a stationary phase for column chromatography. In some embodiments, provided herein is a chromatography kit, comprising the chromatography system of any of the preceding embodiments, and a stationary phase for column chromatography. In some embodiments, provided herein is a chromatography kit, comprising the jacket member of any of the preceding embodiments, a chromatography column, and a stationary phase for column chromatography.

[0069] In some embodiments, provided herein is a chromatography column or chromatography column set, comprising the housing assembly or the housing assembly set of any of the preceding embodiments, and a stationary phase for column chromatography in the internal cavity of one or more 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 column 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 column comprises a stationary phase for column chromatography. In some embodiments, the plurality of chromatography columns can be arranged sequentially or in parallel, optionally wherein the plurality of chromatography columns are operably connected. In some embodiments, the plurality of chromatography columns are operably connected. In any of the preceding embodiments, the plurality of chromatograph columns can comprise a first chromatography column and a second chromatography column, wherein the 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 may comprise an affinity chromatography matrix. In any of the preceding embodiments, the stationary phase may be or comprise a non-magnetic material, a non-ferromagnetic material, or non-paramagnetic material. In any of the preceding embodiments, the stationary phase may be or comprises one selected from the group consisting of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxy ethyl aspartamide) silica, poly(N-isopropylacrylamide) grafted silica, a styrenedivinylbenzene gel, a copolymer of an acrylate or an acrylamide and a diol, a co-polymer of a polysaccharide and N,N'-methylenebisacrylamide, and a combination thereof. In any of the preceding 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 a mean particle size of about 5 µm to about 200 µm, of about 5 µm to about 600 µm, or of about 5 µm to about 1500 µm. In any of the preceding embodiments, the stationary phase may have a mean pore size of about 1 nm to about 500 nm.

[0070] In any of the preceding embodiments, the stationary phase may comprise a selection agent immobilized thereon. In some embodiments, the selection agent is capable of specific binding to a selection marker on the surface of one or more cells. In any of the preceding embodiments, the one or more cells may be or comprise immune cells. In some embodiments, the one or more cells are T cells.

[0071] In any of the preceding embodiments, the selection agent can be or comprise an agent selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof. In any of the preceding embodiments, the selection agent can be or comprise an antibody fragment. In any of the preceding embodiments, the selection agent can be or comprise a Fab fragment. In any of the preceding embodiments, the selection agent can be or comprise one selected from the group of divalent antibody fragments consisting of F(ab') 2 fragments and divalent single-chain Fv (scFv) fragments. In any of the preceding embodiments, the selection agent can be or comprise a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFvs. In any of the preceding embodiments, 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 a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins, and avimers.

[0072] In any of the preceding embodiments, the selection agent can further comprise biotin, a biotin analog that reversibly binds to a streptavidin or avidin, a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody binding the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody binding the oligohistidine tag. In any of the preceding embodiments, the selection agent can comprise a streptavidin-binding peptide.

[0073] In any of the preceding embodiments, the selection marker can be or comprise a T cell coreceptor. In any of the preceding embodiments, the selection marker can be or comprise a member of a T cell antigen receptor complex. In any of the preceding embodiments, the selection marker can be or comprise a CD3 complex. In any of the preceding embodiments, the selection marker can be or comprise a CD3 chain. In any of the preceding embodiments, the selection marker can be or comprise a CD3γ, CD3δ, CD3ε, or CD3ζ chain. In any of the preceding embodiments, the selection marker can be or comprise CD8. In any of the preceding embodiments, the selection marker can be or comprise CD4. In any of the preceding embodiments, the selection marker can be or comprise CD45RA. In any of the preceding embodiments, the selection marker can be or comprise CD27. In any of the preceding embodiments, the selection marker can be or comprise CD28. In any of the preceding embodiments, the selection marker can be or comprise CCR7.

[0074] In any of the preceding embodiments, the specific binding between the selection agent and the selection marker may not result in the induction of a signal, e.g., the induction of a stimulatory or activating or proliferative signal, to the T cells.

[0075] In any of the preceding embodiments, the selection agent can be or comprise an anti-CD3 Fab, an anti-CD8 Fab, or an anti-CD4 Fab. In any of the preceding embodiments, the selection agent can be or comprise an anti-CD27 Fab. In any of the preceding embodiments, the selection agent can be directly or indirectly bound to the stationary phase. In any of the preceding embodiments, the selection agent can be bound indirectly to the stationary phase through a selection reagent to which the selection agent reversibly binds.

[0076] In any of the preceding embodiments, the selection reagent can be or comprise streptavidin, avidin, a mutein of streptavidin that reversibly binds biotin, a biotin analog or a biologically active fragment thereof; a mutein of avidin or streptavidin that reversibly binds a streptavidin-binding peptide; a reagent that comprises at least two chelating groups K, where the 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 a 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 preceding embodiments, the selection reagent can be or comprise a mutein of streptavidin that reversibly binds a streptavidin-binding peptide.

[0077] In any of the preceeding embodiments, the selection agent 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), and the selection agent immobilized on the stationary phase of at least another one of the plurality of chromatography columns can be an anti-CD8 antibody (e.g. an anti-CD8 Fab). In any of the preceeding embodiments, the selection agent immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD4 Fab, and the selection agent immobilized on the stationary phase of at least another one of the plurality of chromatography columns can be an anti-CD8 Fab.

[0078] In any of the preceeding embodiments, the selection agent immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD3 antibody (e.g. an anti-CD3 Fab) and the selection agent immobilized on the stationary phase of at least another one of the plurality of chromatography coluns 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 preceeding embodiments, the selection agent immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD3 Fab and the selection agent immobilized on the stationary phase of at least another one of the plurality of chromatography coluns can be an anti-CD27 Fab).

[0079] In any of the preceeding embodiments, the selection agent immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD4 antibody (e.g. anti-CD4 Fab), the selection agent immobilized on at least another of the plurality of chromatography columns can be an anti-CD8 antibody (e.g. anti-CD8 Fab), and the selection agent immobilized on the stationary phase of at least a further one of the plurality of chromatography coluns 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 preceeding embodiments, the selection agent immobilized on the stationary phase of at least one of the plurality of chromatography columns can be an anti-CD4 Fab, the selection agent immobilized on at least another of the plurality of chromatography columns can be an anti-CD8 Fab, and the selection agent immobilized on the stationary phase of at least a further one of the plurality of chromatography coluns can be an anti-CD27 Fab).

[0080] In any of the preceding embodiments, the chromatography kit, chromatography column, or chromatography column set may further comprise one or more stimulatory agent capable of delivering a stimulatory signal in one or more T cells. In some embodiments, the stationary phase comprises at least one of the one or more stimulatory agent. In some embodiments, the one or more stimulatory agent is immobilized on the stationary phase of the chromatography column or chromatography column set. In some embodiments, the one or more stimulatory agent is indirectly immobilized. In some embodiments, the one or more stimulatory agent is indirectly immobilized via a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide. In some embodiments, the chromatography kit can comprise a stimulatory reagent, wherein the stimulatory reagent comprises one or more stimulatory agent capable of delivering a stimulatory signal in one or more T cells. In some embodiments, the at least one or at least one of the one or more stimulatory agent is a first stimulatory agent, and the chromatography kit, chromatography column, or chromatography column set further comprises one or more second stimulatory agent capable of enhancing, dampening, or modifying the stimulatory signal of the first stimulatory agent. In some embodiments, at least one of the second stimulatory agent is capable of specifically binding to a costimulatory molecule on the one or more T cells, e.g., CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In any of the preceding embodiments, the stationary phase may comprise at least one of the one or more second stimulatory agent.

[0081] In any of the preceding embodiments, the stimulatory signal may be through 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.

[0082] In any of the preceding embodiments, the one or more stimulatory agent can be or comprise an agent selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof. In any of the preceding embodiments, the one or more stimulatory agent can be or comprise an antibody fragment. In any of the preceding embodiments, the one or more stimulatory agent can be or comprise a Fab fragment. In any of the preceding embodiments, the one or more stimulatory agent can be or comprise one selected from the group of divalent antibody fragments consisting of F(ab') 2 fragments and divalent single-chain Fv (scFv) fragments. In any of the preceding embodiments, the one or more stimulatory agent can be or comprise a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFvs. In any of the preceding embodiments, the one or more stimulatory agent can be or comprise a proteinaceous binding molecule with antibody-like binding properties, selected from the group consisting of aptamers, muteins based on a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins, and avimers.

[0083] In any of the preceding embodiments, the one or more stimulatory agent can further comprise biotin, a biotin analog that reversibly binds to a streptavidin or avidin, a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody binding the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody binding the oligohistidine tag.

[0084] In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise an agent selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof. In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise an antibody fragment. In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise a Fab fragment. In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise one selected from the group of divalent antibody fragments consisting of F(ab') 2 fragments and divalent single-chain Fv (scFv) fragments. In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFvs. In any of the preceding embodiments, the first and second stimulatory agents, independently, can be or comprise a proteinaceous binding molecule with antibody-like binding properties, selected from the group consisting of aptamers, muteins based on a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, 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.

[0085] In any of the preceding embodiments, the first and second stimulatory agents, independently, can further comprise biotin, a biotin analog that reversibly binds to a streptavidin or avidin, a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody binding the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody binding the oligohistidine tag. In any of the preceding embodiments, the first and second stimulatory agents, independently, can further comprise a streptavidin-binding peptide.

[0086] In any of the preceding embodiments, the streptavidin-binding peptide can be selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

[0087] In any of the preceding embodiments, the first stimulatory agent and the second stimulatory agent can be reversibly bound to an oligomeric stimulatory reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the size of the oligomeric stimulatory reagent comprises i) a radius of greater than 50 nm, ii) a molecular weight of at least 5 × 10 6< g / mol; and / or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric stimulatory reagent.

[0088] In any of the preceding embodiments, the stimulatory reagent can comprise a plurality of streptavidin or streptavidin mutein molecules, wherein the size of the stimulatory reagent comprises i) a radius of greater than 50 nm, ii) a molecular weight of at least 5 × 10 6< g / mol; and / or (iii) at least 100 streptavidin or streptavidin mutein tetramers per stimulatory reagent.

[0089] In any of the preceding embodiments, the streptavidin mutein can comprise the amino acid sequence Val 44< -Thr 45< -Ala 46< -Arg 47< at sequence positions corresponding to positions 44 to 47 of SEQ ID NO: 1, or the streptavidin mutein can comprise the amino acid sequence lle 44< -Gly 45< -Ala 46< -Arg 47< at sequence positions corresponding to positions 44 to 47 of SEQ ID NO: 1. In any of the preceding embodiments, the N-terminal amino acid residue of the streptavidin mutein can be in 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 in the region of amino acids 133 to 142 of SEQ ID NO: 1. In any of the preceding embodiments, the streptavidin mutein can comprise the amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27, 28, 104, and 105.

[0090] In some embodiments, disclosed herein is a device, comprising the housing assembly, the housing assembly set, or the chromatography kit, chromatography column, or chromatography column set of any of the preceding embodiments, and 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, and 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 house a stationary phase for column chromatography, and the device further comprises an input composition reservoir operably connected to an inlet of the internal cavity to permit intake of an input composition comprised in the input composition reservoir into the internal cavity. In some embodiments, the input composition comprises 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 white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the apheresis or leukapheresis product is freshly isolated from a subject or thawed from a cryopreserved apheresis or leukapheresis product.

[0091] In any of the preceding embodiments, the device can further comprise an output composition reservoir operably connected to the internal cavity via an outlet of the outlet housing member. In any of the preceding embodiments, the device can further comprise an output composition reservoir operably connected to the an outlet of the internal cavity to permit or effect discharge of an output composition comprised in the output composition reservoir from the internal cavity. In some embodiments, the output composition comprises or is enriched T cells. In some embodiments, the enriched T cells have undergone stimulation during chromatography on the chromatography column. In any of the preceding embodiments, the device can be in a closed or sterile system.

[0092] In some embodiments, disclosed herein is a method of preparing a chromatography column or chromatography column set, comprising introducing a stationary phase into the housing assembly or the housing assembly set of any of the preceding embodiments.

[0093] In some embodiments, disclosed herein is a method of preparing a chromatography column or chromatography column set, comprising introducing the stationary phase of the chromatography kit of any of the preceding embodiments into the housing assembly or housing assembly set of the chromatography kit.

[0094] In some embodiments, the competition agent or free binding agent can be an agent that competes for binding with a streptavidin binding peptide of the selection agent to a streptavidin mutein immobilized on the stationary phase. In some embodiments, the competition agent or free binding agent can be a biotin or a biotin analog, optionally wherein the biotin analog is D-biotin. In some embodiments, the competition agent or free binding agent can be D-biotin.

[0095] In any of the preceding embodiments, the stimulatory agent may be or comprise an oligomeric stimulatory reagent comprising (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulatory agent capable of delivering a stimulatory signal in one or more T cells, where the size of the oligomeric stimulatory reagent comprises i) a radius of greater than 50 nm, ii) a molecular weight of at least 5 × 10 6< g / mol; and / or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric stimulatory reagent. In some embodiments, the streptavidin mutein comprises the amino acid sequence Val 44< -Thr 45< -Ala 46< -Arg 47< or lle 44< -Gly 45< -Ala 46< -Arg 47< at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO:1; or the streptavidin mutein comprises the amino acid sequence Val 44< -Thr 45< -Ala 46< -Arg 47< at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1.

[0096] In any of the preceding embodiments, at least one of the one or more stimulatory agent can be capable of delivering a stimulatory signal, wherein the stimulatory signal is through 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.

[0097] In any of the preceding embodiments, the at least one of the one or more stimulatory agent can be a first stimulatory agent capable of delivering the stimulatory signal and the one or more stimulatory agent can further comprise one or more of a second stimulatory agent capable of enhancing, dampening, or modifying the stimulatory signal of the first stimulatory agent. In some embodiments, the second stimulatory agent 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 preceding embodiments, the second stimulatory agent can be capable of specifically binding to CD28 and / or the costimulatory molecule is CD28.

[0098] In any of the preceding embodiments, the first stimulatory agent can specifically bind CD3 and the second stimulatory agent can specifically bind CD28. In any of the preceding embodiments, the first stimulatory agent can comprise a monovalent antibody fragment that binds to CD3 and the second stimulatory agent 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 preceding embodiments, the first stimulatory agent can be an anti-CD3 Fab and the second stimulatory agent can be an anti-CD28 Fab.

[0099] In any of the preceding embodiments, during at least a portion of the incubation, the temperature control member can regulate the temperature of the stationary phase to a target temperature between about 30°C and about 39°C

[0100] In any of the preceding 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 between about 35°C and about 39°C.

[0101] In any of the preceding embodiments, during at least a portion of the incubation, the temperature control member can maintain the temperature of the stationary phase at a target temperature between about 30°C and about 39°C.

[0102] In any of the preceding embodiments, during at least a portion of the incubation, the temperature control member can maintain the temperature of the stationary phase at a target temperature between about 35°C and about 39°C.

[0103] In any of the preceding embodiments, the target temperature is between about 30°C and about 39°C, optionally at or about 37°C. In any of the preceding embodiments, the target temperature can be 37°C or about 37°C.

[0104] In any of the preceding embodiments, during at least a portion of the incubation, the connector may allow intake of gas into the internal cavity. In some embodiments, the gas is sterile and is or comprises air. In any of the preceding embodiments, the intake of gas into the internal cavity can be intermittent or continuous during the incubation.

[0105] In any of the preceding embodiments, the sample may be or 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 white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the apheresis or leukapheresis product is freshly isolated from a subject. In some embodiments, the apheresis or leukapheresis product is thawed from a cryopreserved apheresis or leukapheresis product.Brief Description of the Drawings

[0106] FIGS. 1A and 1B provide a schematic representation of an exemplary housing assembly for column chromatography. FIG. 1A shows the exemplary housing assembly comprising a temperature control member comprising a heating coil with inlet and outlet for external warm water supply, and a gas supply connector for screw-on air filters. FIG. 1B shows the exemplary housing assembly in an exemplary column chromatography system. FIG. 2 provides a schematic representation of an exemplary embodiment for stimulating and selecting for target cells, in which the stimulation is carried out by an incubation of the cells, which occurs, at least in part, in the presence of a support, 36, drawn here as a stationary phase, having immobilized thereon component(s) of a selection reagent 31 for cell selection (Panel A), which 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 with immobilized selection reagent 31, under conditions whereby the selection reagent and selection agent reversibly bind, e.g., via binding sites, generating an oligomeric complex with the selection agent multimerized thereon (Panel B). The selection agent can include more than one agent. Alternatively, the reversibly bound complex of the selection agent and selection reagent may be added to the stationary phase as a complex for immobilization. As shown, cells 33, including target cells, are combined with the stationary phase and multimerized selection agent complex, whereby target cells become reversibly immobilized to the support 36, via the selection agent 32 and reagent (selection marker) 34 (Panel C). Optionally, cells not bound are removed, either prior to addition of stimulatory agents or subsequent thereto. A complex containing multimerized stimulatory agents 35 reversibly bound to an oligomeric stimulatory reagent 37 is added, under conditions whereby the stimulatory agent 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). FIGS. 3A and 3B show results of a WST metabolic assay of T cells from three different donors incubated with anti-CD3 / anti-CD28 multimerized on different batches of oligomeric reagents. FIG. 3A summarizes WST metabolic activity, as indicated by WST ratio, for all tested batches (pooled) compared to reference batches containing anti-CD3 / anti-CD28 multimerized on an oligomeric backbone with an average hydrodynamic radius of 36 nm or 101 nm. The average WST metabolic activity, as indicated by mean WST ratio, among T cells from the different donors for individual tested batches and reference reagents is shown in FIG. 3B. FIG. 4 provides a schematic representation of an exemplary on-column T cell selection and stimulation process. FIG. 5 shows elution efficiency using an exemplary heat / gas column having a heating element and a gas supply element was approximately two-fold of that using the reference column. The estimate (grey bar) was the theoretical number of captured cells that could be eluted assuming 100% efficiency. FIG. 6 shows flow cytometry quantification of cells in the starting material, the negative fraction or the positive fraction, after on-column T cell selection and stimulation using the exemplary column having a heating element and a gas supply element. The cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, CD45 and CD14. FIGS. 7A and 7B show results of T cells after on-column selection and stimulation using the exemplary column having a heating element and a gas supply element. The cells were monitored, at Day 1, Day 2, and Day 3 during the subsequent incubation, for cell number and cell surface expression by flow cytometry after staining the cells with antibodies recognizing CD3, CD4, CD8, and the activation markers CD69 and CD25, and the flow cytometry results are shown in FIG. 7A. Assessment for cell number and fold-expansion following the subsequent incubation showed that the selected and stimulated T cells had started to increase in number at Day 3, as shown in FIG. 7B, consistent with the ability of the cells to proliferate. FIGS. 8A-8C provide results of on-column T cell selection using a cryopreserved apheresis sample as the starting sample, on the exemplary heat / gas column. FIG. 8A shows that cryopreserved apheresis samples (CAPHs) generally have high monocyte content (greater than 20%, as indicated by the % of live CD45+ cells), compared to fresh apheresis samples (APHs). FIG. 8B depicts the percentage of cells positive for CD3 or CD14 in the starting material and positive fraction. The numbers of T cells selected using the chromatography column are shown in FIG. 8C, where two sequential selections for CD3 were carried out. FIG. 9 provides a schematic representation of a selection and stimulation run using two identical exemplary heat / gas columns that were arranged sequentially (Run 1), and a selection and stimulation using two identical exemplary heat / gas columns that were arranged in parallel (Run 2). FIGS. 10A and 10B provide comparisons of results of T cell selection and stimulation in Run 1 and Run 2. FIG. 10A shows flow cytometry analysis of the starting materials, the negative fractions, and the positive fractions, where cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fractions were harvested and incubated, and FIG. 10B, left panel, shows expression of activation markers CD25 and CD69 in the cells at Day 1 in incubation Representative results for cell number in Run 1 (▪) and Run 2 (•) during incubation are shown in FIG. 10B, right panel. FIGS. 11A and 11B provide results of on-column T cell selection using a concentrated blood sample as the starting sample, with CD3 selection and stimulation on two exemplary heat / gas columns arranged in parallel. FIG. 11A shows flow cytometry analysis of the starting material, the negative fraction, and the positive fraction, where cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fraction were harvested and incubated, and CD4 / CD8 and CD25 / CD69 expressions of the incubated cells are shown in FIG. 11B. FIG. 12 provides results of an exemplary process of selecting T cells directly from whole blood, using Sephadex ®< G-50 as the resin in the exemplary heat / gas chromatography column. The starting material, the negative fractions, and the positive fractions from the CD3+ T cell selection were stained with propidium iodine (PI) and a CD3 antibody and quantified by flow cytometry. FIG. 13 shows the effects of 24 hour on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent on CD3, CD4, and CD8 surface expression (assessed as mean fluorescence intensity, MFI) when the respective molecule was used as a selection marker to immobilize the cell on the stationary phase of a chromatography column. Surface expression patterns are compared to control conditions not involving on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent. Cells were isolated from an apheresis sample applied to the stationary phase. FIG. 14 shows exemplary kinetics of downregulation and re-expression of the TCR / CD3 complex upon on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent when CD3 was used as a selection marker to immobilize the cell on the column. Cells were isolated from an apheresis sample applied to the stationary phase. An antibody against the alpha-beta TCR chains was used to assess the the CD3 / TCR complex. FIGS. 15A-15B show phenotypic and functional characteristics of cultured T cells that spontaneously detached during on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent. FIG. 15A shows from left to right T cell size and CD3, CD69, and CD25 expression at 24 hours and 5 days following on-column stimulation. FIG. 15B shows the proliferative capacity of the spontaneously detached cultured T cells, as indicated by cell number and fold expansion. Cells were isolated from an apheresis sample applied to the stationary phase and collected using a wash step. FIGS. 16A-16D show exemplary effects of incubating T cells with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent in the presence or absence of Compound 63 on mTor signaling and viability and growth kinetics. FIG. 16A shows pS6 expression in live CD8+ T cells by memory subset. FIG. 16B shows the mean florescence intensity (mfi) of pS6 expression of total CD8 T cells by treatment as indicated. FIGS. 16C-16D show viability and total T cell numbers, respectively, over time (as indicated by days; d1, etc) in culture after initiation of stimulation ("input"). In FIGS. 16C-16D, black lines correspond to T cell compositions incubated in the presence of Compound 63, and gray lines correspond to T cell compositions incubated in the absence of Compound 63. FIGS. 17A-17F show exemplary functional and phenotypic properties of cryopreserved CAR-T cells generated using methods employing incubation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent in the presence or absence of Compound 63. FIG. 17A shows intracellular expression of Caspase at the time of thaw. FIGS. 17B and 17D show CD8 CAR-T cell and CD4 CAR-T cell phenotypic profiles, respectively, by subset expression of CD27 and / or CCR7. FIGS. 17C and 17E show intracellular IL2, IFNg, or TNF (left panels) or combinations of IL2 and / or IFNg or TNF (right panels) among CD8 CAR-T cells and CD4 CAR-T cells, respectively, stimulated with antigen-bearing targets. FIG. 17F shows expansion and survival over 12 days (left panel) and total expansion metric calculated by area under the growth curve (AUC, right panel) for CAR-T cells stimulated with anti-CAR beads. FIG. 18A shows CD3+, CD4+ and CD8+ T cell yields following cell selection either using the on-column stimulation process or alternative process described in Example 11. FIGS. 18B-18C show the total number of cells ( FIG. 18B) and percentage of live cells ( FIG. 18C) recovered following the use of on-column stimulation or alternative processes described in Example 11. FIGS. 19A-19D show the percentage of live cells (e.g., purity; FIG. 19A), the percentage of live cells expressing the exemplary CAR ( FIG. 19B), the percentage of live cells expressing CD4 at selection and on day 8 of the process ( FIG. 19C), and T cell phenotype distributions (percentage) for each donor ( FIG. 19D) on day 5 in culture (day 8 from the beginning of the process) for the on-column stimulation or the alternative processes described in Example 11. FIG. 20 shows CD19+ HEK cell lysis over time during culture with anti-CD19 CAR T cells engineered using on-column stimulation or alternative processes, as described in Example 11, and under control conditions. FIGS. 21A-21C show antigen-specific CAR T cell IFNg ( FIG. 21A), IL-2 ( FIG. 21B), and TNFα ( FIG. 21C) production for CD4 and CD8 T cells engineered using the on-column stimulation or the alternative processes described in Example 11. FIGS. 22A-22C show the CD4:CD8 ratio ( FIG. 22A), transduction efficiency of engineered T cells (CD4 and CD8 cells combined; FIG. 22B), and the percentage of viable cells ( FIG. 22C) generated using the on-column stimulation or the alternative processes described in Example 11. Three manufacturing runs are shown for each process. FIG. 23 shows tumor size by average radiance across treatment groups 6 days after mice were injected (i.v.) with B cell lymphoma cell line (Raji) and prior to the mice being treated with CAR-T cell compositions. Treatment groups refer to CAR-T cell compositions produced by three manufacturing runs each of the on-column stimulation or the alternative processes described in Example 11. FIG. 24 shows tumor burden in B cell lymphoma cell line (Raji) injected mice over time for each treatment group. CAR T cell treatment effects are shown for on-column stimulation or the alternative processes described in Example 11, and each of the three manufacturing runs (see FIGS. 22A-22C). FIGS. 25-28 provide schematic representations of an exemplary housing assembly for column chromatography. This exemplary housing assembly includes an inlet housing member, an outlet housing member, a side wall member, and a jacket member that surrounds the side wall member as well as portions of the inlet housing member and the outlet housing member. The jacket member of the exemplary housing assembly is made of two jacket components each containing a heating coil with inlet and outlet for external warm water supply. Together, the two jacket components form the jacket member. The exemplary housing assembly also includes a gas supply connector for screw-on air filters (not shown), said gas supply connector connected to an inlet of the inlet housing member. FIG. 25 shows an exploded view of the exemplary housing assembly. FIGS. 26A-26C show views of the interior ( FIG. 26A), side ( FIG. 26B), and exterior ( FIG. 26C) of one jacket component. FIG. 27 shows a view of the exemplary housing assembly such that the inlets for external warm water supply and a portion of an inlet of the inlet housing member are visible. FIG. 28 shows a view of the exemplary housing assembly such that the outlets for external warm water supply and a portion of an outlet of the outlet housing member are visible. Optional features (not shown) for this exemplary housing assembly include a first porous member configured to separate the stationary phase and an inlet of the internal cavity (e.g., a woven polyester mesh), a second porous member configured to separate the stationary phase and an outlet of the internal cavity (e.g., a woven polyester mesh), and tubing set connectors. FIGS. 29-31 provide schematic representations of an exemplary housing assembly for column chromatography. This exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member that surrounds the side wall member as well as portions of the inlet housing member and the outlet housing member. The jacket member of the exemplary housing assembly is made of three jacket components each containing an electric heating element that includes a metal plate. Together, the three jacket components form the jacket member. The exemplary housing assembly also includes a gas supply connector for screw-on air filters (not shown), said gas supply connector connected to an inlet of the inlet housing member. FIG. 29 shows an exploded view of the exemplary housing assembly. FIGS. 30A-30C show three views of one jacket component. FIG. 30D shows the electric heating element. FIG. 31 shows a view of the exemplary housing assembly such that the electrical connections of the electric heating elements as well as a portion of an outlet of the outlet housing member are visible. Optional features (not shown) for this exemplary housing assembly include a first porous member configured to separate the stationary phase and an inlet of the internal cavity (e.g., a woven polyester mesh), a second porous member configured to separate the stationary phase and an outlet of the internal cavity (e.g., a woven polyester mesh), and tubing set connectors. FIG. 32 shows CD27 surface expression of cells after cells were immobilized on the stationary phase of a heated column using CD27 as a selection marker and stimulated on-column with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent. The column was heated using a jacket member containing two heating coils each with inlet and outlet for external warm water supply. The heated column also included a gas supply connector for screw-on air filters. As a control, CD27-selected cells were not subjected to on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent. Cells were isolated from an apheresis sample applied to the stationary phase. FIG. 33 shows CD3 and CD27 surface expression of cells sequentially isolated from an apheresis sample using two separate columns. CD27 was used as a selection marker in the first column, and the positive fraction of the first column was passed to a second column with a CD3 selection marker. Immobilized cells in the second column were stimulated with an anti-CD3 / anti-CD28 oligomeric stimulatory reagent. The second column was heated using a jacket member containing two heating coils each with inlet and outlet for external warm water supply. The heated column also included a gas supply connector for screw-on air filters. FIGS. 34A-34E show CD3+ depletion ( FIG. 34A), CD4 and CD8 expression ( FIG. 34B), CD69 expression ( FIG. 34C), viability ( FIG. 34D), and viable cell number ( FIG. 34E) of cells after on-column stimulation in chromatography columns heated using different heating elements. Columns were heated using jacket members containing two heating coils (water) or three metal plates as electric heating elements (metal). Columns also included a gas supply connector for screw-on air filters. Detailed Description

[0107] In some aspects, provided herein is a housing assembly for column chromatography. In some aspects, the housing assembly for column chromatography comprises 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 house 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 permitting or effecting intake of gas into the internal cavity. In some aspects, provided herein is a housing assembly for column chromatography, comprising: a chromatography column comprising an internal cavity configured to house 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 permitting or effecting intake of gas into the internal cavity. In some embodiments, the chromatography column comprises an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form the internal cavity. In some aspects, the temperature control member comprises one or more heating elements. In some aspects, the housing assembly further comprises a jacket member comprising the temperature control member comprising 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 device comprising 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 examples, for genetic engineering of the cell or cells to manufacture a cell therapy.

[0108] Methods for generating suitable cell populations, e.g., selected (enriched) and stimulated cell populations, for use in cell therapies often require separate selection and stimulation steps which can prolong the manufacturing process. Different reagents and systems are available for generating cell populations suitable for use in cell therapy, such as cells engineered to express recombinant proteins (e.g., chimeric antigen receptors)). However, in some aspects, using these reagents or systems may require a long or a relatively long amount of time to generate the cells, at least in part due to the need to perform multiple processing steps. Multiple processing steps may also result in cellular stress, thus affecting the usefulness of the cells in downstream processing. Furthermore, selection techniques may involve steps that contaminate selected cells with selection-related particles, such as, for example, selection agents such as Fab fragments and competition reagents and / or free binding agents used to facilitate detachment of the cells from the stationary phase, thus requiring additional wash steps and / or media exchange to purify the output composition. The additional processing steps may result in cell stress, potentially affecting downstream cell processing or even cell biology, in addition to requiring considerable time to complete. Additional devices and methods for generating cell compositions are needed.

[0109] In some aspects, provided herein are devices and methods of using the devices for selecting cells from a sample comprising target cells (e.g., T cells, such as CD3+, CD4+, or CD8+ T cells) and / or stimulating the selected cells. In some aspects, the device comprises 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 house 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 permitting or effecting intake of gas into the internal cavity. In some aspects, the stationary phase is configured to immobilize said target cells thereon. In some aspects, the temperature control member comprises one or more heating elements. In some aspects, the device further comprises a jacket member comprising the temperature control member comprising the 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 jacket members for use in selecting cells from a sample comprising target cells (e.g., T cells, such as CD3+, CD4+, or CD8+ T cells) and / or stimulating the selected cells. In some aspects, the jacket member comprises a temperature control member configured to provide heat to a 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 comprises one or more heating elements.

[0110] In one aspect, it is found herein that the methods of on-column selection and / or stimulation of the target cells are improved when the temperature of cells immobilized on the stationary phase in the internal cavity of the column chromatography is controlled to maintain the termperature at at or about 37°C or 37°C ±about 5°C. It also is found herein that a column configuration that permits gas exchange (e.g. presence of air in the column) also improves the overall health, fitness or condition of the cells during the on-column selection and / or stimulation. In particular embodiments, the provided devices are able to the control the temperature (e.g., 37°C or 37°C about 5°C) in the column during the selection and stimulation of cells by the provided on-column methods. In particular embodiments, the provided devices able to control the temperature (e.g., 37°C or 37°C about 5°C) and permit gas exchange, e.g. the presence of air, in the column during the selection and stimulation of cells by the provided on-column methods. In some aspects, the provided device can be used in connection with methods for selection and / or stimulation of cells to facilitate or improve cell activation and downstream processing of the cells, such as detachment or elution of cells from the stationary phase for subsequent genentic engineering of the cells.

[0111] In one aspect, the temperature control member is configured to provide a temperature appropriate for selection and / or stimulation of the target cells immobilized on the stationary phase in the internal cavity of the column chromatography. For this purpose, a heating and / or cooling means can be provided in the device or in a system comprising the device. 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 to 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 target cells immobilized on the stationary phase at an optimal temperature for stimulation by a a stimulatory reagent. In some embodiments, the optimal temperature for stimulation is higher than about 2°C, higher than about 4°C, higher than about 8°C, higher than about 12°C, higher than about 16°C, higher than about 20°C, higher than about 24°C, higher than about 28°C, higher than about 32°C, or higher than about 36°C. In some embodiments, the optimal temperature for stimulation is or is about 37°C. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept at a constant temperature value (e.g., the optimal temperature) during at least portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept 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 portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept 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 portion of the stimulation.

[0112] In one aspect, the device comprises a connector configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas into the internal cavity. Ine one aspect, 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 comprises air.

[0113] In some aspects, the devices and methods, which are not in accordance with the claimed invention, provided herein reduce and / or minimize cell handling and processing time in a manufacturing process. In some aspects, the device comprises 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 comprises one or more member, such as a heating member and / or a gas supply member, that facilitates or promotes cell activation, thereby facilitating or promoting spontaneous detachment of selected and stimulated cells from the stationary phase. In some aspects, the device further comprises one or more member configured to collect the selected and stimulated cells that spontaneously detach from the stationary phase (e.g., due to the stimulation) without the use of a competition agent or a free binding agent to facilitate detachment. In some aspects, the devices and methods (methods are not in accordance with the claimed invention) provided herein are capable of combining cell selection, stimulation, and / or collection steps. In some aspects, the devices and methods provided herein do not require separate steps to facilitate detachment of the selected and stimulated cells from the stationary phase. In some aspects, the devices and methods (methods are not in accordance with the claimed invention) provided herein do not require separate purification steps, e.g., steps to remove agents (e.g., competition agents and / or free binding agents) used to facilitate detachment. As such, the devices and methods (methods are not in accordance with the claimed invention) provided herein reduce the number of processing steps needed to generate a selected and stimulated cell composition suitable for downstream processing (e.g., genetic engineering, expansion, subsequent incubation, stimulation and / or selection (e.g., polishing)), thereby reducing manufacturing time, minimizing potential cell stress, and / or decreasing the potential for contamination. In particular embodiments, the devices and methods (methods are not in accordance with the claimed invention) herein are capable of generating an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within 24 hours.

[0114] The provided devices and methods (methods are not in accordance with the claimed invention) are capable of selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, such as from 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 selected and stimulated cells in the absence of processing steps to detach the cells from the stationary phase and remove agents used to facilitate said detachment from the output composition of selected and stimulated cells. In particular aspects, the provided devices and methods (methods are not in accordance with the claimed invention) are capable of generating populations of selected and stimulated cells in a shortened amount of time compared to methods that include separate selecting and stimulating steps and require additional steps to detach cells from the stationary phase and remove agents used to facilitate detachment. In certain aspects, the provided devices and methods (methods are not in accordance with the claimed invention) are capable of generating a selected and stimulated cell output population (also referred to as a composition) suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection), within 24 hours of initiating stimulation on the column, also referred to herein as on-column stimulation.

[0115] If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications, the definition set forth herein prevails over the definition in the patents, applications, published applications and other publications.

[0116] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DEVICES AND KITS FOR CELL SELECTION, STIMULATION, AND / OR ENGINEERING

[0117] In particular aspects, the provided devices and methods herein are capable of selecting and stimulating target cells (e.g., CD3+, CD4+, or CD8+ T cells) on a stationary phase of a chromatography column, where stimulation facilitates downregulation of the molecule used for cell selection (i.e., selection marker), resulting in spontaneous detachment of the cell from the stationary phase. In some embodiments, the stationary phase of the chromatography column is functionalized with an agent (e.g., selection agent) capable of specifically binding to a molecule (e.g., selection marker) on a target cell surface. In this way, when combining a sample comprising target cells containing the selection marker (e.g., CD3, CD4, CD8) with the stationary phase, 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 Section II-B-1 and II-B-2. In particular aspects, the target cells (e.g., T cells) are stimulated while immobilized on the stationary phase (e.g., on-column stimulation), for example, by addition of stimulatory agents, stimulatory reagents comprising stimulatory agents, and / or via stimulatory agents coupled directly or indirectly to the stationary phase. Exemplary stimulatory agents and stimulatory reagents comprising stimulatory agents (e.g., oligomeric stimulatory 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 condense multiple processing steps (e.g., selection and stimulation) and allow the condensed process to occur within the same container and / or closed system, which can provide increased efficiency and sterility.

[0118] In certain aspects, the provided devices and methods herein involve the use of oligomeric stimulatory reagents comprising stimulatory agents capable of delivering a stimulatory signal to a target cell (e.g., T cell). Existing reagents for use in stimulating T cells in vitro, such as in the absence of exogenous growth factors or low amounts of exogenous growth factors, are known (see e.g. US Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). In general, such reagents may employ beads, e.g., magnetic beads, of greater than 1 µm in diameter to which various binding agents (e.g. anti-CD3 antibody and / or anti-CD28 antibody) are immobilized. However, in some cases, such magnetic beads are, for example, difficult to integrate into methods for stimulating cells under conditions required for clinical trials or therapeutic purposes since it has to be made sure that these magnetic beads are completely removed before administering the expanded T cells to a subject. In some aspects, such removal, such as by exposing the cells to a magnetic field, may decrease the yield of viable cells available for the cell therapy. In certain cases, such reagents, e.g., stimulatory reagents containing magnetic beads, must be incubated with the cells for a minimal amount of time to allow a sufficient amount of detachment of the T cells from the stimulatory reagent. Furthermore, reagents such as beads are not readily compatible with column chromatography due to physical constraints.

[0119] The provided devices and methods herein utilizing oligomeric stimulatory reagents overcome such potential limitations. For example, in some embodiments, the provided methods include addition of a soluble oligomeric reagent not bound to a solid support (e.g., bead) to the stationary phase to initiate stimulation. In some embodiments, the risk of residual reagent output cells generated or produced by the methods is reduced or avoided by use of the oligomeric reagent since addition of a competition reagent or free binding agent can be used to dissociate (e.g., disrupt binding) the oligomeric stimulatory reagents comprising the stimulatory agents from the cells. In some embodiments, this also means that a process that is compliant with GMP standards can be more easily established compared to other methods, such as those where additional measures have to be taken to ensure that the final population for administration is free of beads. Thus, in some aspects, removal or separation of oligomeric stimulatory reagent from cells, such as by the addition of a competition agent or free binding agent, results in little or no cell loss as compared to removal or separation of bead based stimulatory reagents. In some aspects, the timing of the stimulatory reagent or oligomeric stimulatory reagent removal or separation is not limited or is less limited than the removal or separation of bead based stimulatory reagents. Thus, in some aspects, the stimulatory reagent or oligomeric stimulatory reagent may be removed or separated from the cells at any time or step during the provided methods.

[0120] 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 of on-column selection and / or stimulation of target cells. In some aspects, the provided devices allow for the regulation of the temperature, e.g., heating, of cells immobilized on a stationary phase. In some aspects, the provided devices allow for the maintenance of the temperature of the immobilized cells, e.g., at or about 37°C or 37°C about 5°C. In some aspects, the regulation and maintenance of 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 permit gas exchange, e.g., the presence of air in the stationary phase. In some aspects, gas exchange as permitted 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 methods of on-column selection and / or stimulation for use in conjunction with the provided devices provide for improved, e.g., healthier, cells for subsequent engineering for use in a therapy, e.g. an autologous cell therapy.

[0121] In some embodiments, the devices provided herein can be used to perform any of the methods described in Section II.

[0122] In particular aspects, the durations of the provided methods can be measured from when cells, e.g., T cells of an input cell population or sample, are first contacted or exposed to stimulating conditions (e.g., as described herein such as in Section II-D), referred to herein alternatively as the initiation of incubation with a stimulatory agent or under stimulating conditions, e.g., as in when the exposing to the stimulatory reagent is initiated. In some embodiments, the duration of time 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 initiation of incubation (e.g., adding a stimulatory reagent or exposing to a stimulatory reagent). In particular embodiments, the duration of the incubation is, is about, or is 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, is about, or is less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of alternative or existing processes.

[0123] It is contemplated herein that the output compositions of selected and stimulated cells may be further processed. For example, the output cells may be genetically engineered to express a recombinant protein, such as a chimeric antigen receptor, and / or the output cells may undergo further incubation, stimulation, expansion, selection (e.g., polishing), and / or formulation.

[0124] In certain embodiments, methods of using the provided devices are performed on samples, such as, for example, apheresis, buffy coat, or whole blood. In some embodiments, the samples are biological samples. In some embodiments, the biological samples are collected from human subjects. In some embodiments, the biological samples are collect from patients suffering from a disease or condition. In some embodiments, the methods are performed on populations of cells, e.g., CD4+ and CD8+ T cells, that were previously isolated, enriched, or selected from a sample. In some embodiments, the sample or cells isolated from the sample may have been cryopreserved.

[0125] In some embodiments, provided herein are devices, kits, systems, and / or articles of manufacture for cell selection, stimulation, and / or engineering. In some embodiments, provided is an arrangement of a stationary phase for chromatography. In some embodiments, the arrangement further comprises a bioreactor. The bioreactor is suitable for the expansion of cells, and the stationary phase is suitable for cell separation and on-column stimulation. In embodiments, the stationary phase is a gel filtration matrix and / or affinity chromatography matrix, wherein the gel filtration and / or affinity chromatography matrix comprises an selection reagent, wherein the selection reagent comprises a binding site Z1 specifically binding to a binding partner C1 comprised in a selection agent and / or the selection reagent comprises a binding site Z2 specifically binding to a binding partner C2 comprised in a second selection agent. The stationary phase is thereby suitable for immobilizing thereon the first selection agent and / or the second selection agent, the first binding partner C1 and / or the second binding partner C2. In addition the bioreactor and the stationary phase are fluidly connected. This arrangement can be used in a serial expansion and can be integrated into known cell expansion systems such as the Quantum ®< cell expansion system) or the Xuri Cell Expansion System W25.

[0126] In some embodiments, the stationary phase is comprised in a chromatography column. The arrangement may further comprise a second stationary phase which is fluidly connected to the first stationary phase. The secondary stationary phase may be a gel filtration matrix and / or affinity chromatography matrix, wherein the gel filtration and / or affinity chromatography matrix comprises a selection reagent, thereby being suitable of immobilizing the multimerization reagent on the stationary phase. This type of arrangement may facilitate sequential selection of target cells (e.g., T cells, CD4, CD3, CD8 T cells) wherein one of the columns is also suitable for on-column stimulation as described herein.

[0127] Provided embodiments in some aspects are directed to a device for purification (e.g. selection) and culture, such as stimulation or expansion, of a composition of cells, in which the device comprises at least one arrangement of a bioreactor and a first stationary phase or a second stationary phase for chromatography as defined above.

[0128] The device may further comprise a plurality of arrangements of a bioreactor and a stationary phase being fluidly connected in series.

[0129] The device may comprise a sample inlet being fluidly connected stationary phase for chromatography. The device may also comprise a sample outlet for purified and stimulated target cells, the sample outlet being fluidly connected to the stationary phase of the last of the at least one arrangement of a bioreactor and the stationary phase for chromatography.

[0130] In some embodiments, the device may be designed as a functionally closed system.A. Chromatography Housing Assembly

[0131] In some embodiments, provided herein is a chromatography housing assembly (also referred to herein as a housing assembly for column chromatography or a housing assembly) suitable for the chromatography-based cell selection and / or stimulation methods disclosed herein. The chromatography housing assembly may be provided with or without a stationary phase for chromatography.

[0132] In one aspect, provided 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 house 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 permitting or effecting intake of gas into the internal cavity. In one aspect, the housing assembly for column chromatography further comprises a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form the internal cavity. The connector can be disposed on the inlet housing member, the outlet housing member, and / or the side wall member. The connector can be a bonded connector, a screw 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 preceding embodiments, the connector can be configured to sealingly engage tubing in fluid communication with the gas source. In any of the preceding embodiments, the connector can comprise one or more filter, and / or the connector can be operably connected to tubing comprising one or more filter. The one or more filter is a gas filter, e.g., an air filter. The one or more filter can be a sterile filter and / or a sterilizing filter for sterilization by filtration. In one aspect, 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 comprises 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, such as detachment or elution of cells from the stationary phase and / or genetic engineering of the cells.

[0133] In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume between or between about 1 and 40 mL, such as between or between about 1 and 35 mL, 1 and 30 mL, 1 and 25 mL, 1 and 20 mL, 1 and 15 mL, 1 and 10 mL, 1 and 5 mL, 5 and 40 mL, 5 and 35 mL, 5 and 30 mL, 5 and 25 mL, 5 and 20 mL, 5 and 15 mL, 5 and 10 mL, 10 and 40 mL, 10 and 35 mL, 10 and 30 mL, 10 and 25 mL, 10 and 20 mL, 10 and 15 mL, 15 and 40 mL, 15 and 35 mL, 15 and 30 mL, 15 and 25 mL, 15 and 20 mL, 20 and 40 mL, 20 and 35 mL, 20 and 30 mL, 20 and 25 mL, 25 and 40 mL, 25 and 35 mL, 25 and 30 mL, 30 and 40 mL, 30 and 35 mL, or 35 and 40 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume between or between about 15 and 25 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume between or between about 15 and 20 mL. In some embodiments, the internal cavity of the housing assembly can accommodate a bed volume between or between about 18 and 20 mL.

[0134] In some embodiments, the housing assembly for column chromatography includes one or more connectors, e.g., two or more connectors. In some embodiments, the 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 at least on each of the inlet housing member and the outlet housing member. In some embodiments, both the inlet housing member and the outlet housing member have a connector disposed thereon.

[0135] In any of the preceding embodiments, the temperature control member can be configured to regulate or maintain a temperature of the stationary phase in the internal cavity. In any of the preceding 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 between about 35°C and about 39°C (e.g., at or at about 37°C). In some embodiments, the temperature control member can be configured to heat the stationary phase to a target temperature between about 30°C and 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 higher than about 36°C. In some embodiments, the temperature for stimulation is or is about 37°C. In some embodiments, the target temperature (e.g., an optimal temperature for cell stimulation) is higher than about 2°C, at or higher than about 4°C, at or higher than about 8°C, at or higher than about 12°C, at or higher than about 16°C, at or higher than about 20°C, at or higher than about 24°C, at or higher than about 28°C, at or higher than about 32°C, at or higher than about 36°C, at or higher than about 37°C, at or higher than about 38°C, at or higher than about 39°C, or at or higher than about 40°C. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept at a constant temperature value (e.g., an optimal temperature) during at least portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept at a selected temperature value (e.g., an 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 portion of the stimulation. In some embodiments, the temperature of target cells immobilized on the stationary phase is kept 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 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, such as detachment or elution of cells from the stationary phase and / or genetic engineering of the cells. In some aspects, stimulation of (e.g., lymphocytes such as T cells) at an optimal temperature (e.g., 37°C or 37°C ±about 5°C) preserves or maintains the health of the cells during on-column stimulation.

[0136] 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) and in the presence of air (e.g., air) in the column facilitates cell activation and downstream processing of the cells, such as detachment or elution of cells from the stationary phase and / or genetic engineering of the cells.

[0137] FIGS. 1A-1B provide an exemplary housing assembly for column chromatography. In some aspects, housing assembly 1 comprises inlet housing member 2 and outlet housing member 3, and at least the inlet housing member and the outlet housing member form an internal cavity configured to house a stationary phase, such as resin 4 for column chromatography. In some aspects, the housing assembly further comprises a temperature control member, e.g., a temperature control member comprising heating coil 5, configured to provide heat to the stationary phase in the internal cavity. In some aspects, the housing assembly further comprises a connector, e.g., gas exchange connector 6, configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas 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 inlet housing member 2 and outlet housing member 3 have gas exchange connectors 6 disposed thereon.

[0138] In some embodiments, the housing assembly further comprises a side wall member. For example, as shown in FIG. 1A, inlet housing member 2, outlet housing member 3, and side wall member 7 together form the internal cavity.

[0139] 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 side wall member. In some embodiments, both the inlet housing member and the side wall member have a connector disposed thereon. In some embodiments, both the outlet housing member and the side wall member have a connector disposed thereon. In some embodiments, each of the inlet housing member, the outlet housing member, and the side wall 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 side wall member has at least two connectors disposed thereon.

[0140] In some embodiments, the connector is formed between any two or among all three of the inlet housing member, the outlet housing member, and the side wall member. In some aspects, the connector is formed between the inlet housing member and the outlet housing member. In some aspects, the connector is formed between the inlet housing member and the side wall member. In some aspects, the connector is formed between the outlet housing member and the side wall member. In some aspects, at least one connector is formed between the inlet housing member and the side wall member, and at least one connector is formed between the outlet housing member and the side wall member.

[0141] In any of the preceding embodiments, the housing assembly can comprise a plurality of the connectors, e.g., gas exchange connector 6, configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas into the internal cavity. In some embodiments, at least one of the connectors is operably connected to the gas source (directly or indirectly through tubing optionally including one or more filter and / or one or more valve), while at least one other connector is configured to vent.

[0142] In any of the preceding embodiments, the connector can be a bonded connector, a screw 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 preceding embodiments, the connector can comprises a male fitting or a female fitting. In any of the preceding embodiments, the connector can be configured to sealingly engage tubing in fluid communication with the gas source. In any of the preceding embodiments, the connector can comprise one or more valve. In any of the preceding embodiments, the connector can be operably connected to tubing comprising one or more valve. In any of the preceding embodiments, the connector can comprises one or more filter. In any of the preceding embodiments, the connector can be operably connected to tubing comprising one or more filter. In any of the preceding embodiments, the one or more filter can be a gas filter, e.g., an air filter. In any of the preceding embodiments, the one or more filter can be a sterile filter and / or a sterilizing filter for sterilization by filtration.

[0143] In some aspects, the housing assembly comprising an inlet housing member that comprises an upper lid. In some embodiments, the upper lid is removably attached to the inlet housing member or the side wall member. In some embodiments, the upper lid is integrally formed with the inlet housing member or the side wall member. In some embodiments, the connector is disposed on the upper lid.

[0144] In any of the preceding embodiments, the inlet housing member can comprise one or more inlet operably connected to the internal cavity to permit intake of an input composition into the internal cavity. For example as shown in FIG. 1A, inlet housing member 2 comprises an inlet, e.g., tubing set connector 8, disposed on the upper lid. In some embodiments, the connector and the one or more inlet are disposed on the upper lid at different locations, e.g., as shown in FIG. 1A (lower panel) and FIG. 1B. In some embodiments, the connector and the one or more inlet are disposed on the upper lid at the same location. For example, the one or more inlet may be configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas into the internal cavity, while also configured to operably connect to the internal cavity to permit intake of an input composition into the internal cavity. The one or more inlet may be controllably open or close at certain time points during chromatography for intake of gas, and controllably open or close at other time points during chromatography for intake of the input composition.

[0145] In some embodiments, fluid path through the one or more inlet is at an angle of about 90 degrees to the upper lid, while fluid path through the connector is at an angle of about 45 degrees to the upper lid.

[0146] In any of the preceding embodiments, the outlet housing member can comprise a lower lid of the housing assembly. In some aspects, the lower lid is removably attached to the outlet housing member or the side wall member. In other aspects, the lower lid is integrally formed with the outlet housing member or the side wall member.

[0147] In any of the preceding embodiments, the outlet housing member can comprise one or more outlet operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity. In some aspects, the one or more outlet is disposed on the lower lid. In some embodiments, the connector and the one or more outlet are disposed on the lower lid at different locations, e.g., as shown in FIG. 1A (lower panel). In some embodiments, the connector and the one or more outlet are disposed on the lower lid at the same location. For example, the one or more outlet may be configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas into the internal cavity, while also configured to operably connect to the internal cavity to permit or effect discharge of an output composition from the internal cavity. The one or more outlet may be controllably open or close at certain time points during chromatography for intake of gas, and controllably open or close at other time points during chromatography for discharge of the output composition from the internal cavity. In some embodiments, fluid path through the one or more outlet is at an angle of about 90 degrees to the lower lid.

[0148] In any of the preceding embodiments, the gas source can be or comprise a gas reservoir or an outside environment. In any of the preceding embodiments, gas in the gas source can be sterile. In any of the preceding embodiments, the gas can be or comprise air.

[0149] In any of the preceding embodiments, the housing assembly can further comprise tubing operably connected to the gas source. In some embodiments, the tubing is configured to sterilely connect the internal cavity to the gas source. In any of the preceding embodiments, the tubing can comprise one or more valve. In any of the preceding embodiments, the tubing can comprise one or more filter.

[0150] In any of the preceding embodiments, the housing assembly can further comprise one or more porous member, e.g., a cell strainer or a cell sieve. For example, as shown in FIG. 1A (upper panel), housing assembly 1 comprises woven polyester mesh 9. In some embodiments, the housing assembly comprises a first porous member, e.g., woven polyester mesh 9 between inlet housing member 2 and side wall member 7, configured to separate the stationary phase and an inlet of the internal cavity. In some embodiments, the housing assembly further comprises a second porous member, e.g., woven polyester mesh 9 between outlet housing member 3 and side wall member 7, configured to separate the stationary phase and an outlet of the internal cavity.

[0151] In any of the preceding embodiments, the one or more porous member can have an average pore diameter of about 20 µm. In any of the preceding embodiments, the one or more porous member can comprises a mesh having a mesh size of about 20 µm.

[0152] In any of the preceding embodiments, the temperature control member can be configured to regulate or maintain a 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 between about 35°C and about 39°C (e.g., at or at 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.

[0153] In any of the preceding embodiments, the housing assembly can further comprise 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 the power source.

[0154] In any of the preceding embodiments, the temperature control member can comprise a heating source. Alternatively, in any of the preceding embodiments, the temperature control member can be configured to operably connect to a heating source which is external to the housing assembly.

[0155] In some embodiments, the temperature control member includes a heating element. In some embodiments, the heating element is configured to uniformly heat the stationary phase.

[0156] In any of the preceding 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 one aspect, the heating element 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 one aspect, the heating element 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 the stationary phase in the internal cavity. In one aspect, the heating element is a non-electric heating element.

[0157] In some embodiments, the non-electric heating element comprises a heating channel comprising an inlet and an outlet for a heated fluid, e.g., a heated liquid or gas. In some embodiments, the heating channel is a heating coil and the heated fluid is heated water. For example, as shown in FIG. 1A, the housing assembly comprises heating coil inlet 10 and heating coil outlet 11. In some embodiments, the inlet for heated water is configured to connect to an external reservoir of heated water.

[0158] In some embodiments, the heating element is an electric heating element. In some embodiments, the electric heating element 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 the power source.

[0159] In some embodiments, the electric heating element includes a metal plate. In some embodiments, the metal plate is made at least in part of a heat-conductive metal, e.g, aluminum or copper. In some embodiments, the metal plate is made at least in part of aluminum, e.g., made entirely of aluminum. In some embodiments, the electric heating element further includes 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 other components of the electric heating element. In some embodiments, the electrical isolation layer lines at least a portion of one face of the metal plate, e.g., entirely lines one face of the metal plate.

[0160] In some embodiments, at least a portion of the heating element is in contact, e.g., direct 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. In some embodiments, at least a portion of the heating element is in contact, e.g., direct contact, with at least a portion of the side wall member. In some embodiments, at least a portion of the heating element is in contact, e.g., direct contact, with at least a portion of the inlet housing member. In some embodiments, at least a portion of the heating element is in contact, e.g., direct contact, with at least a portion of the outlet housing member.

[0161] In some embodiments, the heating element is in contact, e.g., direct contact, with at least a portion of the inlet housing member, at least a portion of the outlet housing member, and / or the side wall member. In some embodiments, the heating element is in contact, e.g., direct contact, with at least a portion of the side wall member. In some embodiments, the heating element is in contact, e.g., direct contact, with the side wall member.

[0162] 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 side wall 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 side wall member. In some embodiments, the heating element is not in contact with the inlet housing member, the outlet housing member, or the side wall member.

[0163] In any of the preceding embodiments, the heating element can be disposed along and / or around a 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 side wall member, outside the side wall member, or partially inside and partially outside the side wall member.

[0164] In some embodiments, the heating element is disposed inside the internal cavity. In some embodiments, the heating element includes a non-electric heating element disposed inside the internal cavity. In some embodiments, the heating element includes a heating channel disposed inside the internal cavity. In some embodiments, the heating channel includes an inlet and an outlet for a heated fluid, e.g., heated water. In some embodiments, the inlet for the heated water is configured to connect to an external reservoir of heated water.

[0165] In some embodiments, the heating element is disposed outside the internal cavity. In some embodiments, the heating element is disposed outside the side wall 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 side wall member. In some embodiments, the heating element surrounds, e.g., entirely surrounds, the side wall 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 inlet of the inlet housing member, e.g., one or more inlet operably connected to the internal cavity to permit intake of an input composition into the internal cavity, is exposed by the heating element. In some embodiments, at least a portion of one or more inlet of the inlet housing member operably connected to the internal cavity to permit intake of an input composition into the internal cavity is exposed by the heating element. In some embodiments, at least a portion of one of the one or more inlet 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 outlet of the outlet housing member, e.g. one or more outlet operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity, is exposed by the heating element.. In some embodiments, at least a portion of one or more outlet operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity is exposed by the heating element. In some embodiments, at least a portion of one of the one or more outlet of the outlet housing member is outside the heating element.

[0166] In some embodiments, the heating element includes a heating channel surrounding at least a portion of the inlet housing member, the outlet housing member, and / or the side wall member.

[0167] In any of the preceding embodiments, the heating element can comprise a coil surrounding the inlet housing member, the outlet housing member, and / or the side wall member. In any of the preceding embodiments, the heating element can comprise a heating channel surrounding the inlet housing member, the outlet housing member, and / or the side wall member. In any of the preceding embodiments, the heating element can comprise a heating channel surrounding the side wall member.

[0168] In some embodiments, the heating element surrounds at least a portion of the side wall 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 comprises an insulation 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 side wall member. In some embodiments, the insulation 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 side wall member. In some embodiments, the insulation layer surrounds at least a portion of the side wall member, e.g., entirely surrounds the side wall member. In some embodiments, the insulation layer is a solid layer. In some embodiments, the insulation layer is a liquid layer. In some embodiments, the insulation layer is a gas layer. In some embodiments, the insulation layer is an air layer.

[0169] In some embodiments, the temperature control member includes a plurality of heating elements. In some embodiments, the temperature control member includes between or between about 2 and 10 heating elements, between or between about 2 and 8 heating elements, between or between about 2 and 6 heating elements, or between or between about 2 and 4 heating elements, each inclusive. In some embodiments, the temperature control member includes two heating elements. In some embodiments, the temperature control member includes three heating elements. In some embodiments, the temperature control member includes four heating elements.

[0170] In some embodiments, the plurality of heating elements are configured to uniformly heat the stationary phase. In some embodiments, the plurality of heating elements are arranged to uniformly heat the stationary phase.

[0171] In some embodiments, the plurality of 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. In some embodiments, the plurality of heating elements are identical. In some embodiments, the plurality of heating elements are a combination of different heating elements.

[0172] In some embodiments, the plurality of heating elements include a plurality of non-electric heating elements. In some embodiments, the plurality of heating elements include a plurality of heating channels. In some embodiments, each of the plurality of heating channels has an inlet and outlet for a heated fluid, e.g., heated water. In some embodiments, at least two of the plurality of heating channels are fluidly coupled to one another. In some embodiments, the plurality of heating channels are fluidly coupled to one another. 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.

[0173] In some embodiments, the plurality of heating elements include a plurality of electric heating elements, e.g., electric heating elements comprising metal plates. In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to one another. In some embodiments, the plurality of electric heating elements are electrically coupled to one another. 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 or 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 or included in the housing assembly.

[0174] In some embodiments, at least a portion of at least one of the plurality of heating elements is in contact, e.g., direct 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. In some embodiments, at least a portion of at least one of the plurality of heating elements is in contact, e.g., direct contact, with 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 is in contact, e.g., direct contact, with 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 is in contact, e.g., direct contact, with at least a portion of the side wall member.

[0175] In some embodiments, at least a portion of at least one of the plurality of heating elements is in contact, e.g., direct 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. In some embodiments, at least one of the plurality of heating elements is in contact, e.g., direct 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 the side wall member. In some embodiments, at least one of the plurality of heating elements is in contact, e.g., direct contact, with at least a portion of the side wall member. In some embodiments, at least one of the plurality of heating elements is in contact, e.g., direct contact, with the side wall member.

[0176] 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 the side wall 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 side wall 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 side wall member. In some embodiments, the plurality of heating elements is not in contact with the inlet housing member, the outlet housing member, or the side wall member.

[0177] In some embodiments, at least one of the plurality of heating elements is disposed along and / or around a 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 the side wall member, outside the side wall member, or partially inside and partially outside the side wall 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 are disposed inside the internal cavity. In some embodiments, the plurality of heating elements are disposed outside the internal cavity. In some embodiments, the plurality of heating elements are disposed outside the side wall member.

[0178] In some embodiments, at least one of the plurality of heating elements is disposed outside the internal cavity. In some embodiments, 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. In some embodiments, at least one of the plurality of heating elements surrounds at least a portion of the side wall member, e.g., entirely surrounds the side wall member. In some embodiments, the plurality of heating elements surrounds at least a portion of the side wall member, e.g., entirely surrounds the side wall member. In some embodiments, at least one of the plurality of heating elements surrounds at least a portion of the inlet housing member. In some embodiments, the plurality of heating elements surrounds at least a portion of the inlet housing member. In some embodiments, at least a portion of one or more inlet of the inlet housing member is exposed by the plurality of heating elements. In some embodiments, at least a portion of one or more inlet of the inlet housing member is outside the plurality of heating elements. In some embodiments, at least one of the plurality of heating elements surrounds at least a portion of the outlet housing member. In some embodiments, the plurality of heating elements surrounds at least a portion of the outlet housing member. In some embodiments, at least a portion of one or more outlet of the outlet housing member is exposed by the plurality of heating elements. In some embodiments, at least a portion of one or more outlet of the outlet housing member is outside the plurality of heating elements.

[0179] In some embodiments, the plurality of heating elements are uniformly or about uniformly distributed around the side wall member. In some embodiments, the plurality of heating elements are uniformly or about uniformly distributed around the circumference of the side wall member.

[0180] In some embodiments, at least one of the plurality of heating elements surrounds at least a portion of the side wall 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 comprises an insulation 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 side wall member. In some embodiments, the insulation 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 side wall member. In some embodiments, the insulation layer surrounds at least a portion of the side wall member, e.g., entirely surrounds the side wall member. In some embodiments, the insulation layer is a liquid layer. In some embodiments, the insulation layer is a gas layer. In some embodiments, the insulation layer is an air layer.

[0181] In some embodiments, the heating element is disposed outside the side wall member, and the housing assembly further includes a jacket member (also referred to herein as a jacket) that includes the heating element. In some embodiments, the jacket member includes the temperature control member that includes the heating element disposed outside the side wall member. In some embodiments, the jacket member is any as described in Section I-C.

[0182] In some embodiments, at least one of the plurality of heating elements is disposed outside the side wall member, and the housing assembly further includes a jacket member that includes the at least one of the plurality of heating elements. In some embodiments, the jacket member includes the temperature control member that includes the at least one of the plurality of heating elements. In some embodiments, the plurality of heating elements are disposed outside the side wall member, and the housing assembly further includes a jacket member that includes the plurality of heating elements. In some embodiments, the jacket member includes the temperature control member that includes the plurality of heating elements.

[0183] 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 side wall 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 side wall member.

[0184] In some embodiments, the jacket member is releasably 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 side wall member. In some embodiments, the jacket member is not releasably connected together.

[0185] In some embodiments, the jacket member is configured to surround at least a portion of the side wall member. In some embodiments, the jacket member is configured to entirely surround the side wall member. In some embodiments, the jacket member surrounds at least a portion of the side wall member, e.g., entirely surrounds the side wall member. In some embodiments, the jacket member entirely surrounds the side wall member. In some embodiments, the jacket member surrounds at least a portion of the inlet housing member. In some embodiments, one or more inlet of the inlet housing member is exposed by the jacket member. In some embodiments, one or more inlet of the inlet housing member operably connected to the internal cavity to permit intake of an input composition into the internal cavity is exposed by the jacket member. In some embodiments, one or more inlet of the inlet housing member is outside 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 outlet of the outlet housing member is exposed by the jacket member. In some embodiments, one or more outlet of the outlet housing member operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity is exposed by the jacket member. In some embodiments, one or more outlet of the outlet housing member is outside the jacket member.

[0186] In some embodiments, the jacket member is in contact, e.g., direct 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. In some embodiments, the jacket member is in contact, e.g., direct contact, with at least a portion of the side wall member. In some embodiments, the jacket member is in contact, e.g., direct contact, with the side wall member.

[0187] In some embodiments, at least a portion of the jacket member 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 side wall member. In some embodiments, at least a portion of the jacket member is not in contact with the inlet housing member, the outlet housing member, or the side wall member. In some embodiments, the jacket member is not in contact with the inlet housing member, the outlet housing member, or the side wall member.

[0188] In some embodiments, the jacket member includes a non-electric heating element, e.g., a heating channel that includes an inlet and an outlet for heated fluid. In some embodiments, the jacket member includes a plurality of non-electric heating elements. In some embodiments, the jacket member includes at least one opening for the inlet for heated fluid. In some embodiments, the jacket member includes at least one opening for the outlet for heated fluid. In some embodiments, the jacket member includes at least two openings for one or more inlets for heated fluid. In some embodiments, the jacket member includes at least two openings for one or more outlets for 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 or included in the housing assembly.

[0189] In some embodiments, the jacket member includes an electric heating element, e.g., an electric heating element that includes a metal plate. In some embodiments, the jacket member includes a plurality of electric heating elements In some embodiments, the jacket member is arranged such that the electric heating element or the plurality of electric heating elements are configured to electrically connect to a power source.

[0190] 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. In some embodiments, the temperature sensor is configured to electrically connect to a power source, e.g., a power source external to or included in the housing assembly.

[0191] In some embodiments, the jacket member includes one or more jacket components. In some embodiments, the one or more jacket components are configured to together 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 side wall member. In some embodiments, the one or more jacket components are configured to be releasably 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 side wall member.

[0192] In some embodiments, the one or more jacket components are configured to surround at least a portion of the side wall member, e.g., entirely surround the side wall member. In some embodiments, the one or more jacket components are configured to entirely surround the side wall 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 inlet of the inlet housing member is exposed by the one or more jacket components. In some embodiments, one or more inlet of the inlet housing member is outside 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 outlet of the outlet housing member is exposed by the one or more jacket components. In some embodiments, one or more outlet of the outlet housing member is outside the one or more jacket components.

[0193] In some embodiments, the jacket member includes two or more jacket components, for instance between or between about 2 and 10 jacket components, 2 and 8 jacket components, 2 and 6 jacket components, or 2 and 4 jacket components, each inclusive. In some embodiments, the jacket member includes two jacket components. In some embodiments, the jacket member includes three jacket components. In some embodiments, the jacket member includes four jacket components.

[0194] In some embodiments, at least two of the two or more jacket components each include a heating element. In some embodiments, the two or more jacket components each include a heating element.

[0195] In some embodiments, at least two of the two or more jacket components each include a temperature sensor. In some embodiments, the two or more jacket components each include a temperature sensor.

[0196] 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 heating channel with inlet and outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include a heating channel with inlet and outlet for a heated fluid, e.g., heated water.

[0197] In some embodiments, the heating channels of the at least two of the two or more jacket components are fluidly coupled to one another. In some embodiments, the heating channels of the two or more jacket components are fluidly coupled to one another.

[0198] In some embodiments, at least one of the two or more jacket components includes an opening for the inlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for the inlet for a fluid, e.g., heated water.

[0199] In some embodiments, at least one inlet of the heating channels of the two or more jacket components is configured to connected 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 connected to an external reservoir of heated fluid.

[0200] In some embodiments, at least one of the two or more jacket components includes an opening for the outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each include an opening for the outlet for a fluid, e.g., heated water.

[0201] FIGS. 25-28 provide schematic representations of an exemplary housing assembly for column chromatography. The exemplary housing assembly 1 shown in FIG. 25 includes inlet housing member 2, outlet housing member 3, and side wall member 7 that form an internal cavity configured to house a stationary phase. Housing assembly 1 also includes a gas supply connector for screw-on air filters (not shown) and a temperature control member that includes heating coils 5, as shown in FIGS. 26A-26C. Heating coils 5 are contained in a jacket member made of two jacket components 12. The jacket components 12 are configured to together entirely surround side wall member 7 and to surround at least a portion of each of inlet housing member 2 and outlet housing member 3. Each jacket component 12 includes an inlet groove 13 such that the jacket member exposes an inlet of the inlet housing member 2. Each jacket component 12 also includes an outlet groove 14 such that the jacket member exposes an outlet of the outlet housing member 3.

[0202] FIGS. 26A-26C show interior, side, and exterior views of jacket component 12. As shown in FIGS. 26A-26C, each jacket component 12 includes a heating coil 5 for a heated fluid, e.g., heated water. The two heating coils 5 of the jacket member are configured to together entirely surround side wall member 7 and to surround at least a portion of each of inlet housing member 2 and 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 inlets 10 are parallel to an inlet of inlet housing member 2. As shown in FIG. 28, the heating coil outlets 1 are parallel to an outlet of outlet housing member 3.

[0203] In some embodiments, at least two of the two or more jacket components each comprise an electric heating element, e.g., an electric heating element that includes a metal plate. In some embodiments, at least two of the two or more jacket components each comprise an electric heating element. In some embodiments, the two or more jacket components each comprise an electric heating element.

[0204] In some embodiments, the electric heating elements of the at least two of the two or more jacket components are electrically coupled to one another. In some embodiments, the electric heating elements of the two or more jacket components are electrically coupled to one another.

[0205] 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 or 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 or included in the housing assembly.

[0206] In some embodiments, at least one electric heating element of the 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 or 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 or included in the housing assembly.

[0207] FIGS. 29-31 provide schematic representations of an exemplary housing assembly for column chromatography. The exemplary housing assembly 1 shown in FIG. 29 includes inlet housing member 2, outlet housing member 3, and side wall member 7 that form an internal cavity configured to house a stationary phase. Housing assembly 1 also includes a gas supply connector for screw-on air filters (not shown) and a temperature control member that includes electric heating elements 17 that include metal plates. Electric heating elements 17 are part of a jacket member made of three jacket components 12. The jacket components 12 are configured to together entirely surround side wall member 7 and to surround at least a portion of each of inlet housing member 2 and outlet housing member 3. Each jacket component 12 includes an inlet groove 13 such that the jacket member exposes an inlet of the inlet housing member 2. Each jacket component 12 also includes an outlet groove 14 such that the jacket member exposes an outlet of the outlet housing member 3.

[0208] FIGS. 30A-30C show three views of 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 heating element electrical connection 16, and each temperature sensor is configured to electrically connect to a power source via temperature sensor electrical connection 20. As shown in FIG. 30D, electric heating element 17 also includes an electric isolation layer 19 and an aluminum profile 21. Each electric heating element 17 is mounted into jacket component 12 at mounting points 15. Electric heating elements 17 and jacket components 12 are configured such that electric heating elements 17 are uniformly distributed around the circumference of side wall member 7. As shown in FIG. 29, heating element electrical connections 16 and temperature sensor electrical connections 20 are exposed on the same face of the jacket member as the outlet of outlet housing member 3.

[0209] In one aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising 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 permitting or effecting intake of sterile gas into the internal cavity.

[0210] In one aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase and comprising 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 permitting or effecting intake of sterile gas into the internal cavity.

[0211] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising a heating element comprising a metal plate 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 permitting or effecting intake of sterile gas into the internal cavity.

[0212] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase, wherein the temperature control member comprises two heating coils configured to provide heat to the stationary phase; a jacket member comprising the temperature control member comprising the two heating coils, wherein the jacket member is releasably connected together to surround at least a portion of the inlet housing member, the outlet housing member, and the side wall member and the two heating coils entirely surround the side wall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby permitting or effecting intake of sterile gas into the internal cavity.

[0213] In another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member configured to regulate or maintain a temperature of the stationary phase, wherein the temperature control member comprises three electric heating elements each comprising a metal plate and that are configured to provide heat to the stationary phase; a jacket member comprising the temperature control member comprising the three electric heating elements, wherein the jacket member is releasably connected together to surround at least a portion of the inlet housing member, the outlet housing member, and the side wall member and the two heating coils entirely surround the side wall member; and a connector configured to operably and sterilely connect the internal cavity to a gas filter, thereby permitting or effecting intake of sterile gas into the internal cavity.

[0214] In yet another aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising a heating element comprising 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 permitting or effecting intake of sterile gas into the internal cavity. In some embodiments, the heating coil comprises 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 side wall member.

[0215] In one aspect, disclosed herein is a housing assembly for column chromatography, comprising: an inlet housing member, an outlet housing member, and a side wall member, wherein the inlet housing member, the outlet housing member, and the side wall member form an internal cavity configured to house a stationary phase for column chromatography; a temperature control member comprising 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 permitting or effecting intake of sterile gas into the internal cavity.

[0216] In any of the preceding embodiments, the gas filter can be an air filter and the sterile gas can be sterile air. In any of the preceding embodiments, the housing assembly can further comprise the gas filter.

[0217] Also disclosed herein is a housing assembly set, comprising a plurality of the housing assembly disclosed herein. The housing assembly set can comprise at least two of the plurality of the housing assembly arranged sequentially. The housing assembly set can comprise at least two of the plurality of the housing assembly arranged in parallel.

[0218] Also disclosed herein is a chromatography system, comprising 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.B. Chromatography Kits, Columns, and Column Sets

[0219] In some embodiments, also disclosed herein is a chromatography kit, comprising the housing assembly or the housing assembly set disclosed herein, and a stationary phase for column chromatography. In some embodiments, the housing assembly or the housing assembly set is any as described in Section I-A. In some embodiments, the chromatography kit further comprises one or more stimulatory agent orstimulatory reagent. In some embodiments, the one or more stimulatory agent or stimulatory reagent is any as described in Section II-B-1 or II-B-2.

[0220] In some embodiments, also disclosed herein is a chromatography column or chromatography column set, comprising the housing assembly or the housing assembly set disclosed herein, and a stationary phase for column chromatography in the internal cavity of one or more of the housing assembly. In some embodiments, the housing assembly or the housing assembly set is any as described in Section I-A.

[0221] In some embodiments, also disclosed herein is a chromatography column, including a jacket member and a chromatography column. In some embodiments, the internal cavity of the chromatography column includes a stationary phase for column chromatography. In some embodiments, the jacket member is any as described in Section I-C.

[0222] In some embodiments, also disclosed herein is a chromatography column set, including at least one jacket member and a plurality of chromatography columns. In some embodiments, the jacket member is any as described in Section I-C. In some embodiments, the internal cavity of each of the plurality of chromatography columns comprises a stationary phase for column chromatography. In some embodiments, the plurality of chromatography columns are arranged sequentially. In some embodiments, the plurality chromatography columns are arranged in parallel. In some embodiments, the plurality of chromatography columns are operably connected.

[0223] In some embodiments, the plurality of chromatography columns includes a first chromatography column. In some embodiments, the plurality of chromatography columns includes a second chromatography column. In some embodiments, the at least one jacket member is configured to surround the second chromatography column.

[0224] In any of the preceding embodiments, the stationary phase can comprise a gel filtration matrix, and / or an affinity chromatography matrix. The stationary phase may comprise a non-magnetic material, a non-ferromagnetic material, or non-paramagnetic material. In other aspects, the stationary phase is selected from the group consisting of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxy ethyl aspartamide) silica, poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of an acrylate or an acrylamide and a diol, a co-polymer of a polysaccharide and N,N'-methylenebisacrylamide, and a combination thereof. The stationary phase can comprises or is a monolithic matrix, a particulate matrix, and / or a planar matrix.

[0225] In any of the preceding embodiments, the particulate matrix can have a mean particle size of about 5 µm to about 200 µm, of about 5 µm to about 600 µm, or of about 5 µm to about 1500 µm. In any of the preceding embodiments, the stationary phase can have a mean pore size of about 1 nm to about 500 nm.

[0226] In any of the preceding embodiments, the stationary phase can comprise immobilized thereon any of the agents described in Section II-B-1. In some embodiments, the agents are directly immobilized on the stationary phase. In some embodiments, the agents are indirectly immobilized on the stationary phase. In some embodiments, the agents are irreversibly immobilized on the stationary phase. In some embodiments, the agents are reversibly immobilized on the stationary phase. In some embodiments, the agents are reversibly immobilized on the stationary phase via a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide. In some embodiments, the streptavidin mutein and / or the streptavidin-binding peptide are any as described in Section II-B-2.

[0227] In any of the preceding embodiments, the stationary phase can comprise a selection agent immobilized thereon. In some aspects, the selection agent is capable of specific 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.

[0228] Also disclosed here in is an apparatus, comprising the housing assembly, the housing assembly set, or the chromatography kit, chromatography column, or chromatography column set, further comprising an input composition reservoir operably connected to the internal cavity via an inlet of the inlet housing member. In some embodiments, the input composition comprises 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 white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the apheresis or leukapheresis product is freshly isolated from a subject or thawed from a cryopreserved apheresis or leukapheresis product. In some embodiments, the apparatus further comprises an output composition reservoir operably connected to the internal cavity via an outlet of the outlet housing member. In some aspects, the output composition comprises or is enriched T cells. In other aspects, the enriched T cells have undergone stimulation during chromatography on the chromatography column. In any of the preceding embodiments, the apparatus can be a closed or sterile system. An exemplary apparatus that includes an exemplary housing assembly 1 is shown in FIG. 1B.

[0229] Also disclosed herein is a method of preparing a chromatography column or chromatography column set, comprising introducing a stationary phase into the housing assembly or the housing assembly set disclosed herein. In addition, disclosed herein is a method of preparing a chromatography column or chromatography column set, comprising introducing the stationary phase of the chromatography kit into the housing assembly or housing assembly set of the chromatography kit.C. Jacket Members for Chromatography

[0230] The devices provided herein also include jacket members for column chromatography. In some aspects, the provided jacket members are devices that allow for improved methods involving the isolation, processing, or manipulation of target cells immobilized on a stationary phase of a chromatography column, e.g., methods of on-column selection and / or stimulation of target cells. In some aspects, the provided jacket members allow for the regulation of the temperature, e.g., heating, of the immobilized cells. In some aspects, the provided jacket members allow for the maintenance of the temperature of the immobilized cells, e.g., at or about 37°C or 37°C ±about 5°C. In some aspects, the regulation and maintenance of the temperature of cells by the provided devices improves on-column manipulation, e.g., stimulation, of immobilized cells, for instance by improving the overall health, fitness, or condition of the immobilized cells during on-column manipulation.

[0231] In one aspect, the jacket member includes one or more jacket components configured to surround at least a portion of a chromatography column. In some aspects, the chromatography column is configured to house 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 a temperature of the stationary phase. In some embodiments, the one or more heating elements are any as described in Section I-A. In some embodiments, the temperature control member is any as described in Section I-A.

[0232] In some aspects, the one or more jacket components are configured to be releasably connected together to surround the at least a portion of the chromatography column. In other embodiments, the one or more jacket components are configured to be not releasably connected together.

[0233] In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column that can accommodate a bed volume between or between about 1 and 40 mL, such as between or between about 1 and 35 mL, 1 and 30 mL, 1 and 25 mL, 1 and 20 mL, 1 and 15 mL, 1 and 10 mL, 1 and 5 mL, 5 and 40 mL, 5 and 35 mL, 5 and 30 mL, 5 and 25 mL, 5 and 20 mL, 5 and 15 mL, 5 and 10 mL, 10 and 40 mL, 10 and 35 mL, 10 and 30 mL, 10 and 25 mL, 10 and 20 mL, 10 and 15 mL, 15 and 40 mL, 15 and 35 mL, 15 and 30 mL, 15 and 25 mL, 15 and 20 mL, 20 and 40 mL, 20 and 35 mL, 20 and 30 mL, 20 and 25 mL, 25 and 40 mL, 25 and 35 mL, 25 and 30 mL, 30 and 40 mL, 30 and 35 mL, or 35 and 40 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column that can accommodate a bed volume between or between about 15 and 25 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column that can accommodate a bed volume between or between about 15 and 20 mL. In some embodiments, the jacket member is configured to surround at least a portion of a chromatography column that can accommodate a bed volume between or between about 18 and 20 mL.

[0234] In some embodiments, at least a portion of the jacket member is configured to be in contact, e.g., direct contact, with at least a portion of chromatography column. In some embodiments, the jacket member is configured to be in contact, e.g., direct contact, with at least a portion of chromatography column. In some embodiments, the jacket member is configured to be in contact, e.g., direct contact, with the chromatography column.

[0235] In some embodiments, at least a portion of the jacket member is configured to not be in contact with at least a portion of the chromatography column. In some embodiments, at least a portion of the jacket member is configured to not be in contact with the chromatography column. In some embodiments, the jacket member is configured to not be in contact with the chromatography column.

[0236] In some embodiments, the jacket member is configured so that an insulation layer can be disposed between the one or more jacket components and at the chromatography column. In some embodiments, the jacket member further includes an insulation layer. In some embodiments, the insulation layer is configured to be disposed between the one or more jacket components and at least a portion of the chromatography column. In some embodiments, the insulation layer is configured to be disposed between the one or more jacket components and the chromatography column. In some embodiments, the insulation layer is configured to surround at least a portion of the chromatography column.

[0237] In some embodiments, the insulation layer includes a gas layer, e.g., an air layer. In some embodiments, the insulation layer includes a liquid layer. In some embodiments, the insulation layer includes a solid layer.

[0238] In some embodiments, the temperature control member can be configured to heat a stationary phase contained in a chromatography column to a target temperature between about 30°C and about 39°C (e.g., at or at 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 higher than about 36°C. In some embodiments, the temperature control member can be configured to heat the stationary phase to or to about 37°C. In some embodiments, the temperature control member can be configured to heat the stationary phase to at or higher than about 2°C, at or higher than about 4°C, at or higher than about 8°C, at or higher than about 12°C, at or higher than about 16°C, at or higher than about 20°C, at or higher than about 24°C, at or higher than about 28°C, at or higher than about 32°C, at or higher than about 36°C, at or higher than about 37°C, at or higher than about 38°C, at or higher than about 39°C, or at or higher than about 40°C. In some embodiments, the temperature control member can be configured to keep the stationary phase at a target temperature. In some embodiments, the temperature control member can be configured to keep 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 keep 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.

[0239] In some embodiments, the temperature control member can be configured to regulate or maintain a temperature of a stationary phase contained in a chromatography column. In some aspects, the temperature control member is configured to heat, e.g., uniformly heat, the stationary phase from a starting temperature (e.g., room temperature) to a target temperature between about 30°C and about 39°C (e.g., at or at about 37°C). In some aspects, the temperature control member is further configured to maintain the stationary phases at the target temperature.

[0240] In some embodiments, the jacket member 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 jacket member. In some embodiments, the jacket member further includes the power source.

[0241] In any of the preceding embodiments, the temperature control member can comprise a heating source. Alternatively, in any of the preceding embodiments, the temperature control member can be configured to operably connect to a heating source which is external to the housing assembly.

[0242] In some embodiments, the temperature control member includes a heating element. In some embodiments, the heating element is configured to uniformly heat the stationary phase.

[0243] In any of the preceding 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 one aspect, the heating element 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 one aspect, the heating element 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 the stationary phase in the internal cavity. In one aspect, the heating element is a non-electric heating element.

[0244] In some embodiments, the non-electric heating element comprises a heating channel comprising an inlet and an outlet for a heated fluid, e.g., a heated liquid or gas. In some embodiments, the heating channel is a heating coil. In some embodiments, the heated fluid is heated water. In some embodiments, the inlet for heated water is configured to connect to an external reservoir of heated water.

[0245] In some embodiments, the heating element is an electric heating element. In some embodiments, the electric heating element 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 the power source.

[0246] In some embodiments, the electric heating element includes a metal plate. In some embodiments, the metal plate is made at least in part of a heat-conductive metal, e.g, aluminum or copper. In some embodiments, the metal plate is made at least in part of aluminum, e.g., made entirely of aluminum. In some embodiments, the electric heating element further includes an electrical isolation layer, e.g., between at least a portion of the metal plate and at least a portion of other components of the electric heating element. In some embodiments, the electrical isolation layer lines at least a portion of one face of the metal plate, e.g., entirely lines one face of the metal plate.

[0247] In some embodiments, at least a portion of the heating element is configured to be in contact, e.g., direct contact, with at least a portion of the chromatography column. In some embodiments, the heating element configured to be in contact, e.g., direct contact, with at least a portion of the chromatography column. In some embodiments, the heating element configured to be in contact, e.g., direct contact, with the chromatography column.

[0248] In some embodiments, at least a portion of the heating element is configured to be not in contact with at least a portion of the chromatography column. In some embodiments, the heating element is configured to be not in contact with at least a portion of the chromatography column. In some embodiments, the heating element is configured to be not in contact with the chromatography column.

[0249] In some embodiments, the heating element is configured to surround at least a portion of the chromatography column.

[0250] In some embodiments, the temperature control member includes a plurality of heating elements. In some embodiments, the temperature control member includes between or between about 2 and 10 heating elements, between or between about 2 and 8 heating elements, between or between about 2 and 6 heating elements, or between or between about 2 and 4 heating elements, each inclusive. In some embodiments, the temperature control member includes two heating elements. In some embodiments, the temperature control member includes three heating elements.

[0251] In some embodiments, the plurality of heating elements are configured to uniformly heat the stationary phase.

[0252] In some embodiments, the plurality of 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. In some embodiments, the plurality of heating elements are identical. In some embodiments, the plurality of heating elements are a combination of different heating elements.

[0253] In some embodiments, the plurality of heating elements include a plurality of non-electric heating elements. In some embodiments, the plurality of heating elements include a plurality of heating channels. In some embodiments, each of the plurality of heating channels has an inlet and outlet for a heated fluid, e.g., heated water. In some embodiments, at least two of the plurality of heating channels are fluidly coupled to one another. In some embodiments, the plurality of heating channels are fluidly coupled to one another. 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 liquid. In some embodiments, the inlet of each of the plurality of heating channels is configured to connect to an external reservoir of heated liquid.

[0254] In some embodiments, the plurality of heating elements include a plurality of electric heating elements, e.g., electric heating elements comprising metal plates. In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to one another. In some embodiments, the plurality of electric heating elements are electrically coupled to one another. 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 or 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 or included in the housing assembly.

[0255] In some embodiments, at least a portion of at least one of the plurality of heating elements is configured to be in contact, e.g., 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 be in contact, e.g., 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 be in contact, e.g., direct contact, with the chromatography column. In some embodiments, the plurality of heating elements is configured to be in contact, e.g., direct contact, with the chromatography column.

[0256] In some embodiments, at least a portion of at least one of the plurality of heating elements is configured to be not in 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 not in contact with the chromatography column. In some embodiments, at least one of the plurality of heating elements is configured to be not in contact with the chromatography column. In some embodiments, the plurality of heating elements is configured to be not in contact with the chromatography column.

[0257] In some embodiments, the plurality of heating elements are configured to be uniformly or about uniformly distributed around the chromatography column, e.g., around the circumference of the chromatography column.

[0258] In some embodiments, the temperature control member includes a non-electric heating element, e.g., a heating channel for heated fluid. In some embodiments, the temperature control member includes a plurality of non-electric heating elements. In some embodiments, the jacket member includes at least opening for one inlet and at least one opening for one outlet for heated fluid, e.g., heated water. In some embodiments, the jacket member includes at least two openings for inlets and / or at least two openings for outlets for 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 or included in the housing assembly.

[0259] In some embodiments, the jacket member includes an electric heating element, e.g., an electric heating element that includes a metal plate. In some embodiments, the jacket member includes a plurality of electric heating elements. In some embodiments, the electric heating element is 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.

[0260] In some embodiments, at least two of the plurality of electric heating elements are electrically coupled to one another. In some embodiments, the plurality of electric heating elements are electrically coupled to one another.

[0261] In some embodiments, the jacket member includes two or more jacket components, for instance between or between about 2 and 10 jacket components, 2 and 8 jacket components, 2 and 6 jacket components, or 2 and 4 jacket components, each inclusive. In some embodiments, the jacket member includes two jacket components. In some embodiments, the jacket member includes three jacket components. In some embodiments, the jacket member includes four jacket components.

[0262] 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.

[0263] 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.

[0264] In some embodiments, at least two of the two or more jacket components each comprise a non-electric heating element. In some embodiments, at least two of the two or more jacket components each comprise a heating channel with inlet and outlet for a heated fluid, e.g., heated water. In some embodiments, the two or more jacket components each comprise a heating channel with inlet and outlet for a heated fluid, e.g., heated water.

[0265] In some embodiments, the heating channels of the at least two of the two or more jacket components are fluidly coupled to one another. In some embodiments, the heating channels of the two or more jacket components are fluidly coupled to one another.

[0266] In some embodiments, at least one of the two or more jacket components includes an opening for the 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.

[0267] In some embodiments, at least one of the two or more jacket components includes an opening for the 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.II. METHODS FOR SELECTING, STIMULATING, AND / OR ENGINEERING CELLS (not part of the claimed invention)

[0268] The methods disclosed in the following part II are not in accordance with the claimed invention. Using a device disclosed herein, provided herein are methods for generating an output population of cells (also referred to as an output composition), such as selected and stimulated CD3+ T, CD4+ T, and / or CD8+ T cells, including steps for the selection, stimulation, and collection of the cells. In certain embodiments, the methods provided herein are used in connection with manufacturing, generating, or producing a cell therapy. In some embodiments, the methods of generating or producing the output composition, e.g., selected and stimulated T cells, include one or more of steps for isolating cells from a subject, incubating the cells under stimulatory conditions, and genetically engineering the cells. In some embodiments, the method includes processing steps carried out in an order in which input cells, e.g. primary CD4+ and CD8+ T cells, are isolated, such as selected or separated, from a biological sample and incubated under stimulating conditions and collected in a single step, and subsequently genetically engineered to introduce a recombinant polynucleotide encoding a recombinant receptor into the cells, such as by transduction or transfection; and then collected, harvested, or filled into a container, e.g., a bag or vial, as an output population. In some embodiments, the cells of the output population are re-introduced into the same subject, optionally after cryopreserving and storing the cells. In some embodiments, the output populations of engineered cells are suitable for use in a therapy, e.g., an autologous cell therapy.

[0269] Using a device disclosed herein, provided herein are methods for selecting cells from a sample comprising target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells) and immobilizing said target cells on the stationary phase of a chromatography column, stimulating 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 the use of competition agents or free binding agents to facilitate detachment. Among the provided methods are methods involving selecting cells from a sample comprising target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells) and immobilizing said target cells on the stationary phase of a chromatography column, stimulating 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 a stationary phase of a chromatography column, facilitates downregulation of the molecule used for cell selection (i.e., selection marker), resulting in spontaneous detachment or release of the cell from the stationary phase. The release or detachment of the cells can occur without any additional steps or reagents. In some aspects, the cells can be collected by gravity flow, such as by adding a media or other solution to the chromatography column. In particular embodiments, the media or other solution that is added does not contain a competition agents or free binding agents to facilitate detachment of the cells from the stationary phase.

[0270] In particular embodiments, the provided methods are carried out to select and stimulate T cells. In some embodiments, the T cells are selected from a biological sample, e.g. apheresis sample, by adding cells of the sample to an affinity chromatography matrix (e.g. stationary phase) immobilized with or bound by a selection agent specific for T cells or a subset thereof, e.g. as described in Section II.B-1. In provided embodiments, the methods include stimulating the cells immobilized on the stationary phase in the presence of one or more stimulatory agents of the T cells. In some embodiments, the one or more stimulatory agents include an agent for delivering a stimulatory signal in the T cells. In some embodiments, the stimulatory signal is through 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. In some embodiments, the stimulatory agent (e.g. first stimulatory agent) is an agent that binds to CD3, such as an anti-CD3 antibody. In some embodiments, the one or more stimulatory agent further includes a second stimutory agent that is able to further stimulate or enhance a signal in the T cells. In some embodiments, the second stimulatory agent is capable of specifically binding to a costimulatory molecule on the one or more T cells, e.g., 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 include an anti-CD3 antibody and an anti-CD28 antibody, for example, an anti-CD3 Fab and an anti-CD28 Fab. In some embodiments, the one or more stimulatory agent are immobilized or bound to a reagent (e.g. is a stimulatory reagent) that is added to the chromatography column. In particular embodiments, the stimulatory reagent is soluble polymeric or oligomeric reagent. For instance, the one or more stimulatory agents are functionalized to an oligomeric or polymeric protein as opposed to a solid surface (e.g. bead). Exemplary oligomeric stimulatory reagents for use in the provided methods are described herein, e.g. Section II.B-2. In some embodiments, the oligomeric stimulatory reagents is an oligomeric streptavidin mutein that is functionalized or multimerized with one or more stimulatory agents (e.g. anti-CD3 Fab and anti-CD28 Fab). In provided methods, the selected and stimulated T cells are collected by eluting or washing the selected and stimulated cells by gravity flow.

[0271] In some embodiments, said collecting includes washing the stationary phase with media (e.g. serum free media), the media not containing a competition agent or free binding agent to elute the target cells (e.g. T cells) from the stationary phase. In some embodiments, the collecting by gravity flow includes adding media to the stationary phase, the media not comprising a competition agent or free binding agent to elute the T cells from the stationary phase. In some embodiments, said composition containing stimulated T cells does not contain a competition agent or free binding agent. In some embodiments, said competition agent or free binding agent is or contains biotin or a biotin analog, for example a biotin analog that is D-biotin. In some embodiments, the competition agent or free binding agent is D-biotin. In some embodiments, the media for the washing column to elute the cells by gravity flow is a serum-free media that contain recombinant cytokines (e.g. IL-2,

[0272] In some embodiments, the method further includes introducing a recombinant nucleic acid molecule into the stimulated T cells of the composition, wherein the nucleic acid molecule encodes 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.

[0273] 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 the cells introduced with the recombinant receptor (e.g. transduced T cells). In some embodiments, the further incubation is carried out at or about 37 °C ± 2 °C. In some embodiments, the further incubation is carried out under conditions that do not expand or substantially expand the cells. In some embodiments, the further incubation is carried out under conditions for expansion (e.g. proliferation) of the cells. In some embodiments, the further incubation is carried out in the presence of a further agent that is capable of delivering a signal to T cells. In some embodiments, the further agent is contained in the media used for washing the stationary phase. In some embodiments, the further agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells and / or CD8+ T cells. In some embodiments, the further 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 time that is 72 hours, no more than 48 hours, no more than 24 hours, or no more than 12 hours.

[0274] In particular embodiments, using a device disclosed herein, provided herein are methods in connection with generating an output population of cells expressing a recombinant receptor from an initial or input population of cells. In certain embodiments, the input population is produced, generated, and / or made by combining, mixing, and / or pooling cells including from a population of cells containing enriched T cells, enriched CD4+ T cells, and / or enriched CD8+ T cells (herein after also referred to as populations of enriched T cells, populations of enriched CD4+ T cells, and populations of enriched CD8+ T cells, respectively). In some embodiments, the input population of cells is a population of combined, mixed, and / or pooled CD4+ and CD8+ T cells. In certain embodiments, the provided methods are used in connection with genetically engineering the selected and stimulated cells, e.g., to introduce a polynucleotide encoding a recombinant protein by transduction or transfection. In certain embodiments, the methods may be used to isolate select cells from a biological sample (e.g., whole blood, apheresis) to generate an input population of enriched T cells, such as from a biological sample taken, collected, and / or obtained from a subject. In some embodiments, the provided methods may be used in connection with harvesting, collecting, and / or formulating populations of enriched T cells after the cells have been engineered, transduced, and / or cultured.

[0275] In certain embodiments, using a device disclosed herein, provided herein are methods in connection with introducing a heterologous or recombinant polynucleotide into the cells, e.g., transducing or transfecting the cells, such as by a method described herein, e.g., in Section II-F. In particular embodiments, the cells are incubated either during or after genetically engineering the cells, for example, for an amount of time sufficient to allow for integration of a heterologous or recombinant polynucleotide encoding a recombinant protein or to allow for the expression of the recombinant protein. In certain embodiments, the cells are incubated for a set or fixed amount of time, such as an amount of time greater than 18 hours or less than 4 days. In some embodiments, the engineering step is started or initiated within a set amount of time from when the stimulating is started or initiated, such as within 24 hours from when the cells are exposed to a stimulatory agent.

[0276] In some embodiments, the one or more process steps are carried out, at least in part, in serum free media. In some embodiments, the serum free media is a defined or well-defined cell culture media. In certain embodiments, the serum free media is a controlled culture media that has been processed, e.g., filtered to remove inhibitors and / or growth factors. In some embodiments, the serum free media contains proteins. In certain embodiments, the serum-free media may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors. In some embodiments, the serum free media includes cytokines. In some embodiments, the serum free media includes cytokines or recombinant cytokines. In some embodiments, the serum free media includes recombinant IL-2, IL-15, and / or IL-7. In some embodiments, the serum free media includes glutamine. In some embodiments, the serum free media includes glutamine and recombinant IL-2, IL-15, and IL-7.

[0277] In some embodiments, using a device disclosed herein, provided herein are methods that are carried out such that one, more, or all steps in the preparation of cells for clinical use, e.g., in adoptive cell therapy, are carried out without exposing the cells to non-sterile conditions. In some embodiments, the cells are selected, stimulated, transduced, washed, and formulated, all within a closed, sterile system or device. In some embodiments, the one or more of the steps are carried out apart from the closed system or device. In some such embodiments, the cells are transferred apart from the closed system or device under sterile conditions, such as by sterile transfer to a separate closed system.

[0278] In some embodiments, the methods provided herein are performed using any of the devices described in Section I.

[0279] In particular embodiments, the sample and / or isolated portions of the sample (e.g., buffy coat, populations of enriched T cells) may be collected, formulated for cryoprotection, frozen (e.g.,cryoprotected), and / or stored below 0°C, below -20°C, or at or below -70C or - 80°C prior to, during, or after any stage or step of the methods as provided herein. In some embodiments, the cells may be stored for an amount of time under 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or an amount of time under 1, 2, 3, 4, 5, 6, 7, 8 weeks, or for an amount of time at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for more than 8 weeks. After storage, the sample or isolated portion of the sample may be thawed and processing according to the method may be resumed from the same point in the process. In particular embodiments, cultivated and / or formulated populations of enriched T cells are cryoprotected and stored prior to being administered to a subject, e.g., as an autologous cell therapy.

[0280] In particular embodiments, at any stage or step in the process, a portion of the cells may be sampled or collected, e.g., cells may be taken from the population of cells (such as a population of T cells) while the population remains in the closed system. In certain embodiments, such cells may be analyzed for makers, features, or characteristics including but not limited to viability, apoptosis, activation, stimulation, growth, and / or exhaustion. In some embodiments, the cells are sampled or collected by an automated process. In some embodiments, the analysis of sampled or collected cells is automated. In particular embodiments, the analysis is performed in a closed system under sterile conditions.

[0281] In some embodiments, cells or populations of cells that are produced and / or processed by the provided methods may be compared to cells or populations of cells processed or produced by an exemplary and / or alternative process. In certain embodiments, the alternative and / or exemplary process may differ in one or more specific aspects, but otherwise contains similar or the same features, aspects, steps, stages, reagents, or conditions of the embodiment or aspect of the provided methods that be compared to an exemplary or alternative process. For example, selected and stimulated cells generated by the provided methods, e.g., an output composition of cells, may be compared to cells that were generated with a process that involved separate selection and stimulating steps which required use of a competition agent or free binding agent to detach the selected cells from a stationary phase. In some embodiments, unless otherwise specified, the provided methods and the exemplary or alternative process would have been otherwise similar and / or identical, such as with similar or identical steps for selecting, enriching, stimulating, engineering, transfecting, transducing, cultivating, and / or formulating. In some embodiments, unless otherwise specified, the provided methods and the alternative process select and / or enrich cells from the same or similar types of biological samples, and / or process cells and / or input cells of the same cell type.

[0282] In some embodiments, the selected and stimulated cells are a composition containing stimulated T cells in which the T cells have been selected from a biological sample (e.g. apheresis or whole blood sample) containing a plurality of T cells. In some embodiments, the collecting and / or eluting of the selected and stimulated cells that spontaneously detach from the stationary phase is accomplished via gravity flow, for example during a wash step. The methods provided herein combine cell selection, stimulation, and collection and / or elution steps, and do not require separate steps to facilitate detachment of the selected and stimulated cells from the stationary phase and purification steps to remove agents (e.g., competition agents and / or free binding agents) used to facilitate detachment. As such, the methods reduce the number of processing steps needed to generate a selected and stimulated cell composition suitable for downstream processing (e.g., genetic engineering, expansion, subsequent incubation, stimulation and / or selection (e.g., initial selection and / or polishing)), thereby reducing manufacturing time, minimizing potential cell stress, and decreasing the potential for contamination.

[0283] In particular embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within 24 hours. In particular embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours. In particular embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within about 6, 5, 4, 3, or 2 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 6 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 5.5 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 5 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 4.5 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 4 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 3 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 3 to 6 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 4 to 6 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 5 to 6 hours. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a set amount of time, such as within or within less than about 4 to 5 hours. In some embodiments, the methods provided herein generate a composition of engineered T cells (e.g., a therapeutic cell composition) within 5 days. In some embodiments, the methods provided herein generate a composition of engineered T cells (e.g., a therapeutic cell composition) in or in about 4 to 5 days. In some embodiments, the steps provided herein result in a manufacturing process that is or is about 4 or 5 days in length. In some embodiments, the steps provided herein result in a manufacturing process that is about 4 to 5 days in length. In some embodiments, the steps provided herein result in a manufacturing process that is or is about 4 days in length or 96 ± 6 hours in length.

[0284] The provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, such as from 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 selected and stimulated cells in the absence of processing steps to detach the cells from the stationary phase and remove agents (e.g., competition agents or free binding agents) used to facilitate said detachment from the output composition of selected and stimulated cells. In particular embodiments, the provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, such as from 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 selected and stimulated cells by gravity flow.

[0285] In particular aspects, the provided methods are improved compared to 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) prior to stimulating cells. In some embodiments, the one or more additional steps present in existing methods can include an elution step or steps with a competition reagent or free binding agent to recover or collect the selected cells and / or steps to remove reagents used in the selection (e.g. magnetic bead reagents or antibodies). In some embodiments, such additional steps can prolong a process for engineering cells for a cell therapy and / or can result in manipulations of cells during the process that may impact their differentiation state, viability or cell number. In particular aspects, the provided methods generate populations of selected and stimulated cells in a shortened amount of time compared to methods that include separate selecting and stimulating steps and require additional steps to detach cells from the stationary phase and remove agents used to facilitate detachment.

[0286] In certain aspects, the methods generate a selected and stimulated cell output population (also referred to as an output composition) suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within 24 hours of initiating stimulation on the column, also referred to herein as on-column stimulation. In some embodiments, the methods generate a selected and stimulated cell output population (e.g., output composition) suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours of initiating stimulation on the column. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 6, 5, 4, 3, or 2 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 3 to 6 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 4 to 6 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 5 to 6 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 4 to 5 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 6 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 5.5 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 5 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 4.5 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 4 hours. In some embodiments, the methods generate a selected and stimulated cell output population suitable for downstream processing (e.g., genetic engineering, expansion, and / or subsequent rounds of incubation, stimulation, and / or selection (e.g., polishing)), within or within about 3 hours.

[0287] In some embodiments, the methods involve the use of stimulatory agents capable of binding to molecules on the surface of the cells, thereby delivering a stimulatory signal to the cell. In some embodiments, the stimulatory agents are comprised in an oligomeric stimulatory reagent (e.g. a streptavidin mutein oligomer conjugated to anti-CD3 and anti-CD28 Fabs) that can be added to the stationary phase. In some embodiments, the stimulation results in the spontaneous detachment of the selected cells from the stationary phase, thus allowing collection and / or elution of the selected and stimulated cells in the absence of additional processing steps to detach the cells from the stationary phase and remove agents used to facilitate said detachment from the output stimulated cell composition. In some embodiments, the stimulation results in the spontaneous detachment or release of the selected cells from the stationary phase, thus allowing collection and / or elution of the selected and stimulated cells by gravity flow. In some embodiments, gravity flow is relied upon to collect or elute the spontaneously detached cells from the column (e.g., stationary phase). In some embodiments, a wash step, for example in combination with gravity flow, may be used to elute the spontaneously detached cells from the column (e.g., stationary phase). In some embodiments, the wash step can simply include adding cell media (e.g. serum free media) to the column, such as the same media present in the cell input composition prior to adding or immobilizing the cells on the stationary phase. In particular aspects, the methods successfully generate an uncontaminated (e.g., free of agents used for detachment (e.g., competition agents, free binding agents) and / or selection agents) composition of selected and stimulated cells suitable for further processing, e.g., genetic engineering, expansion, incubation, or subsequent rounds of stimulation and / or selection (e.g., polishing), within 24 hours of initiating on-column stimulation. Also provided are articles of manufacture and apparatus thereof

[0288] Different methods are available for generating cell populations suitable for use in cell therapy (e.g., selected (enriched) and stimulated cell populations, engineered to express recombinant proteins (e.g., chimeric antigen receptors)). However, in some aspects, these methods may require a long or a relatively long amount of time to generate the cells, at least in part due to the need to perform multiple processing steps. Multiple processing steps may also result in cellular stress, thus affecting the usefulness of the cells in downstream processing. Additional methods for generating cell compositions are needed.

[0289] In particular aspects, the provided methods are based on observations that selecting and stimulating target cells (e.g., CD3+, CD4+, or CD8+ T cells) on a stationary phase of a chromatography column, where stimulation facilitates downregulation of the molecule used for cell selection (i.e., selection marker), results in spontaneous detachment of the cell from the stationary phase. In some embodiments, the stationary phase of the chromatography column is functionalized with an agent (e.g., selection agent) capable of specifically binding to a molecule (e.g., selection marker) on a target cell surface. In this way, when combining a sample comprising target cells containing the selection marker (e.g., CD3, CD4, CD8) with the stationary phase (e.g., adding the sample to the stationary phase), target cells (e.g., CD3+, CD4+, CD8+ T cells) are indirectly immobilized to the stationary phase. In particular aspects, the target cells (e.g., T cells) are stimulated while immobilized on the stationary phase (e.g., on-column stimulation), for example, by addition of stimulatory agents, stimulatory reagents comprising stimulatory agents, and / or via stimulatory agents coupled directly or indirectly to the stationary phase. In particular embodiments, the stimulatory agents include agents that activate or stimulate T cells, such as anti-CD3 / anti-CD28 antibody (e.g. Fab) agents. Thus, in some aspects, the provided methods and other embodiments are advantageous in that they condense multiple processing steps (e.g., selection and stimulation) and / or eliminate processing steps (e.g., steps for removing selection reagents and / or agents used to facilitate detachment) and allow the condensed process to occur within the same container and / or closed system, which can provide increased efficiency and sterility.

[0290] In certain aspects, the methods involve the use of oligomeric stimulatory reagents comprising stimulatory agents capable of delivering a stimulatory signal to a target cell (e.g., T cell). Exemplary oligomeric reagents include streptavidin mutein oligomers that are reversibly bound or conjugated to one or more antibody or fragment thereof capable of delivering a stimulatory signal to a target cell, e.g. a T cell. In some embodiments, the oligomeric stimulatory reagent is a streptavidin mutein oligomer conjugated to anti-CD3 and anti-CD28 Fabs. Existing reagents for use in stimulating T cells in vitro, such as in the absence of exogenous growth factors or low amounts of exogenous growth factors, are known (see e.g. US Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). In general, such reagents may employ beads, e.g., magnetic beads, of greater than 1 µm in diameter to which various binding agents (e.g. anti-CD3 antibody and / or anti-CD28 antibody) are immobilized. However, in some cases, such magnetic beads are, for example, difficult to integrate into methods for stimulating cells under conditions required for clinical trials or therapeutic purposes since it has to be made sure that these magnetic beads are substantially or completely removed before administering the engineered T cells to a subject. In some aspects, such removal, such as by exposing the cells to a magnetic field, may decrease the yield of viable cells available for the cell therapy. In certain cases, such reagents, e.g., stimulatory reagents containing magnetic beads, must be incubated with the cells for a minimal amount of time to allow a sufficient amount of detachment of the T cells from the stimulatory reagent. Furthermore, reagents such as beads are not readily compatible with column chromatography due to physical constraints.

[0291] The provided methods utilizing oligomeric stimulatory reagents (e.g. streptavidin mutein oligomer conjugated to anti-CD3 and anti-CD28 antibodies, such as Fabs) overcome such potential limitations. For example, in some embodiments, the provided methods include addition of a soluble oligomeric reagent not bound to a solid support (e.g., bead) 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 stimulatory reagent that may be present at the end of an overall process of engineering cells for a cell therapy. In some embodiments, the risk of residual reagent in output cells, e.g. engineered cells, generated or produced by the methods is reduced or avoided by use of the oligomeric reagent since addition of a competition reagent or free binding agent can be used to dissociate (e.g., disrupt binding) the oligomeric stimulatory reagents from the stimulatory agents in a composition containing the cells. In some embodiments, it also may be sufficient to reduce or remove the oligomeric stimulatory reagent from cells in a composition by one or more washing steps, such as without the need to add a competition reagent or free binding agent, since the oligomeric stimulatory reagent is soluble. In some embodiments, this also means that a process that is compliant with GMP standards can be more easily established compared to other methods, such as those where additional measures have to be taken to ensure that the final population for administration is free of beads. Thus, in some aspects, removal or separation of oligomeric stimulatory reagent from cells, such as by the addition of a competition agent or free binding agent or by one or more washing steps, results in little or no cell loss as compared to removal or separation of bead based stimulatory reagents. In some aspects, the timing of the stimulatory reagent or oligomeric stimulatory reagent reduction, removal or separation is not limited or is less limited than the removal or separation of bead based stimulatory reagents. Thus, in some aspects, the stimulatory reagent or oligomeric stimulatory reagent may be reduced, removed or separated from the cells at any time or step during the provided methods.

[0292] Also provided are cells and populations prepared by the methods, including pharmaceutical populations and formulations, and kits, systems, and devices for carrying out the methods. Further provided are methods for use of the cells and populations prepared by the methods, including therapeutic methods, such as methods for adoptive cell therapy, and pharmaceutical populations for administration to subjects.A. Samples and Cell Preparation

[0293] In particular embodiments, using a device disclosed herein, provided herein are methods, which are not in accordance with the claimed invention, that include selecting and / or enriching cells from a biological sample. In some embodiments, the provided methods include selecting cells or populations thereof from biological samples, such as those obtained from or derived from a subject, such as one having a particular disease or condition or in need of a cell therapy or to which cell therapy will be administered. In some aspects, the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0294] In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0295] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0296] In some embodiments, the sample is a sample containing T cells. In some embodiments, the sample 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 white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the sample is an apheresis sample. In some embodiments, the sample is a leukaphresis sample.

[0297] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca 2+< / Mg 2+< free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0298] In some embodiments, the sample containing cells (e.g., an apheresis product or a leukapheresis product) is washed in order to remove one or more anti-coagulants, such as heparin, added during apheresis or leukapheresis.

[0299] In some embodiments, the sample containing cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed prior to any steps for isolating, selecting, activating, stimulating, engineering, transducing, transfecting, incubating, culturing, harvesting, formulating a population of the cells, and / or administering the formulated cell population to a subject.

[0300] In particular embodiments, an apheresis product or a leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed before being subject to a cell selection or isolation step (e.g., a T cell selection or isolation step) as described infra. In some embodiments, the thawed cell composition is subjected to dilution (e.g., with a serum-free medium) and / or wash (e.g., with a serum-free medium), which in some cases can remove or reduce unwanted or undesired components. In some cases, the dilution and / or wash removes or reduces the presence of a cryoprotectant, e.g. DMSO, contained in the thawed sample, which otherwise may negatively impact cellular viability, yield, recovery upon extended room temperature exposure. In some embodiments, the dilution and / or wash allows media exchange of a thawed cryopreserved product into a serum-free medium, such as one described herein or in PCT / US2018 / 064627.

[0301] In some embodiments, the serum-free medium comprises a basal medium (e.g.OpTmizer ™< T-Cell Expansion Basal Medium (ThermoFisher), supplemented with one or more supplement. In some embodiments, the one or more supplement is serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion, and / or activation of a cell (e.g., a T cell), such as provided by an additional supplement (e.g. OpTmizer ™< T-Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium further comprises a serum replacement supplement, for example, an immune cell serum replacement, e.g., ThermoFisher, #A2596101, the CTS ™< Immune Cell Serum Replacement, or the immune cell serum replacement described in Smith et al. Clin Transl Immunology. 2015 Jan; 4(1): e31. In some embodiments, the serum-free medium further comprises a free form of an amino acid such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax ™< (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and / or recombinant human IL-15.

[0302] In some embodiments, after a cryopreserved and / or cryoprotected apheresis product or leukapheresis product is subject to a T cell selection or isolation step, no additional cryopreservation and / or cryoprotection step is performed during or between any of the subsequent steps, such as the steps of activating, stimulating, engineering, transducing, transfecting, incubating, culturing, harvesting, formulating a population of the cells, and / or administering the formulated cell population to a subject. For example, T cells selected from a thawed cryopreserved and / or cryoprotected apheresis product or leukapheresis product are not again cryopreserved and / or cryoprotected before being thawed for a downstream process, such as transduction.

[0303] In particular embodiments, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is banked (e.g., without cell selection before freezing the sample), which, in some aspects, can allow more flexibility for subsequent manufacturing steps. In one aspect, banking cells before selection increases cell yields for a downstream process, and banking cells earlier may mean they are healthier and may be easier to meet manufacturing success criteria. In another aspect, once thawed, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product can be subject to one or more different selection methods. Advantages of this approach are, among other things, to enhance the availability, efficacy, and / or other aspects of cells of a cell therapy for treatment of a disease or condition of a subject, such as in the donor of the sample and / or another recipient.

[0304] In some embodiments, the sample (e.g. apheresis or leukapheresis sample) is collected and cryopreserved and / or cryoprotected prior to or without prior cell selection (e.g., without prior T cell selection, such as selection by chromatography), at a time after the donor is diagnosed with a disease or condition. In some aspects, the time of cryopreservation also is before the donor has received one or more of the following: any initial treatment for the disease or condition, any targeted treatment or any treatment labeled for treatment for the disease or condition, or any treatment other than radiation and / or chemotherapy. In some embodiments, the sample is collected after a first relapse of a disease following initial treatment for the disease, and before the donor or subject receives subsequent treatment for the disease. The initial and / or subsequent treatments may be a therapy other than a cell therapy. In some embodiments, the collected cells may be used in a cell therapy following initial and / or subsequent treatments. In one aspect, the cryopreserved and / or cryoprotected sample without prior cell selection may help reduce up-front costs, such as those associated with non-treatment patients in a randomized clinic trial who may crossover and require treatment later.

[0305] In some embodiments, the sample (e.g. apheresis or leukapheresis sample) is collected and cryopreserved and / or cryoprotected prior to or without prior cell selection (e.g., without prior T cell selection, such as selection by chromatography), at a time after a second relapse of a disease following a second line of treatment for the disease, and before the donor or subject receives subsequent treatment for the disease. In some embodiments, patients are identified as being likely to relapse after a second line of treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on disease type and / or genetics, such as double-hit lymphoma, primary refractory cancer, or activated B-cell lymphoma. In some embodiments, the risk factors are based on clinical presentation, such as early relapse after first-line treatment, or other poor prognostic indicators after treatment (e.g., IPI (International Prognostic Index) > 2).

[0306] In some embodiments, the sample (e.g. apheresis or leukapheresis sample) is collected and cryopreserved and / or cryoprotected prior to or without prior cell selection (e.g., without prior T cell selection, such as selection by chromatography), at a time before the donor or subject is diagnosed with a disease. In some aspects, the donor or subject may be determined to be at risk for developing a disease. In some aspects, the donor or subject may be a healthy subject. In certain cases, the donor or subject may elect to bank or store cells without being deemed at risk for developing a disease or being diagnosed with a disease in the event that cell therapy is required at a later stage in life. In some embodiments, a donor or subject may be deemed at risk for developing a disease based on factors such as genetic mutations, genetic abnormalities, genetic disruptions, family history, protein abnormalities (such as deficiencies with protein production and / or processing), and lifestyle choices that may increase the risk of developing a disease. In some embodiments, the cells are collected as a prophylactic.

[0307] In some embodiments, the cryopreserved and / or cryoprotected sample of cells (e.g. apheresis or leukapheresis sample), such as a sample of cells that has not been subjected to a prior cell selection (e.g., without prior T cell selection, such as selection by chromatography) is stored, or banked, for a period of time greater than or equal to 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the sample is stored or banked for a period of time greater than or equal to 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the sample is placed into long-term storage or long-term banking. In some aspects, the sample is stored for a period of time greater than or equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 1 1 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more.

[0308] In some embodiments, an apheresis or leukapheresis sample taken from a donor is shipped in a cooled environment to a storage or processing facility, and / or cryogenically stored at the storage facility or processed at the processing facility. In some embodiments, before shipping, the sample is processed, for example, by selecting T cells, such as CD4+ and / or CD8+ T cells. In some embodiments, such processing is performed after shipping and before cryogenically storing the sample. In some embodiments, the processing is performed after thawing the sample following cryogenical storage.

[0309] By allowing donors to store their cells at a stage when the donors, and thus their cells, have not undergone extensive treatment for a disease and / or prior to contracting of a disease or condition or diagnosis thereof, such cells may have certain advantages for use in cell therapy compared to cells harvested after one or after multiple rounds of treatment. For example, cells harvested before one or more rounds of treatment may be healthier, may exhibit higher levels of certain cellular activities, may grow more rapidly, and / or may be more receptive to genetic manipulation than cells that have undergone several rounds of treatment. Another example of an advantage according to embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before they are needed for cell therapy, the cells would be readily available if and when a recipient later needs them. This could increase apheresis lab capacity, providing technicians with greater flexibility for scheduling the apheresis collection process.

[0310] Exemplary methods and systems for cryogenic storage and processing of cells from a sample, such as an apheresis sample, can include those described in International published application no. WO2018170188. In some embodiments, the method and systems involve collecting apheresis before the patient needs cell therapy, and then subjecting the apheresis sample to cryopreservation for later use in a process for engineering the cells, e.g. T cells, with a recombinant receptor (e.g. CAR). In some cases, such processes can include those described herein. In some embodiments, an apheresis sample is collected from a subject and cryopreserved prior to subsequent T cell selection, activation, stimulation, engineering, transduction, transfection, incubation, culturing, harvest, formulation of a population of the cells, and / or administration of the formulated cell population to a subject. In such examples, the cryopreserved apheresis sample is thawed prior to subjecting the sample to one or more selection steps, such as any as described herein.

[0311] In some embodiments, the cryopreserved and / or cryoprotected sample of cells (e.g. apheresis or leukapheresis sample), such as a sample of cells that has not been subject to a prior cell selection (e.g., without prior T cell selection, such as selection by chromatography) is thawed prior to its use for downstream processes for manufacture of a cell population for cell therapy, for example, a T cell population containing CAR+ T cells. In some embodiments, such a cryopreserved and / or cryoprotected sample of cells (e.g. apheresis or leukapheresis sample) is used in connection with the process provided herein for engineering a T cell therapy, such as a CAR+ T cell therapy. In particular examples, no further step of cryopreservation is carried out prior to or during the harvest / formulation steps.B. Agent and Reagent Systems

[0312] In embodiments, using a device disclosed herein, provided herein are methods, which are not in accordance with the claimed invention, that include selecting and / or enriching cells (e.g. T cells) from a biological sample using an agent that binds to a cell surface markers on cells present in a biological sample (selection agent). In provided embodiments, the biological sample is any as described in Section II.A. In some embodiments, the biological sample is a sample that contains T cells. In provided embodiments, the selection agent is bound or immobilized on a chromatography matrix (e.g. stationary phase) contained in a chromatography column of a device provided herein, and effects specific selection of target cells (e.g. T cells) of interest, as described in Section II.C, thereby immobilizing the target cells (e.g. T cells) to the chromatography matrix (e.g. stationary phase). In some embodiments, the selection agent is capable of being bound indirectly to the chromatography matrix (e.g., stationary phase) through a reagent, e.g., selection reagent. In some embodiments, the selection reagent is bound covalently or non-covalently to the stationary phase of the column. In some embodiments, the selection reagent is a reagent that reversibly immobilizs the selection agent on the chromatography matrix (e.g., stationary phase). Exemplary selection reagents to which a selection agent is bound for use in connection with the provided devices and methods are described in Secion II.B.2.

[0313] In some embodiments, the selection reagent to which the selection agent is bound provides a reversible system in which the selection agent is reversibly associated with the reagent. Exemplary reversible systems for selection of cells by chromatography include those described in WO2013 / 124474. In some embodiments as described further herein, the reversible system employs a reagent composed of streptavidin mutein molecules that reversibly bind to the selection agent via a streptavidin-binding peptide binding partner contained by the selection agent. In some embodiments, adding a free binding partner or competition agent (also called competition substance) disrupts the binding between the selection agent and the reagent, thereby reversing binding of the selection agent from the reagent and releasing the immobilized cells free from the selection reagent. For instance, in the case of a streptavidin mutein / streptavidin binding peptide system an exemplary compeitition agent is biotin or a biotin analog (e.g. D-Biotin).

[0314] In some embodiments, reversibility of the binding of the selection agent on the chromatography matrix is not necessary, since on-column stimulation of cells immobilized on the chromatography matrix as provided herein facilitates downregulation of the molecule used for cell selection (i.e., selection marker), resulting in spontaneous detachment or release of the cell from the stationary phase. Thus, the release or detachment of the cells can occur without any additional steps or reagents. In some aspects, the cells can be collected by gravity flow, such as by adding a media or other solution to the chromatography column. In particular embodiments, the media or other solution that is added does not contain a competition agent or free binding agent to facilitate detachment of the cells from the stationary phase. For instance, in the case of a streptavidin mutein / streptavidin binding peptide system, the release or detachment of cells can occur spontaneously such that the cells can be collected by gravity flow after adding a wash or media to the column in which the wash solution or media does not contain a free binding partner or compeitition agent, such as biotin or a biotin analog (e.g. D-Biotin).

[0315] In embodiments, using a device disclosed herein, provided herein are methods, which are not in accordance with the claimed invention, that include on-column stimulation of cells (e.g. T cells) immobilized on the chromatography column, such as by the selection agent or selection reagent. In provided embodiments, the stimulation is carried out using one or more agent for stimulating cells to bind to one or more receptor molecule on the cell to deliver a signal to cells (one or more stimulatory agent). In some embodiment, the one or more stimulatory agent is for stimulating T cells and provides a primary signal to the T cells (e.g. via TCR complex signaling) and a costimulatory signal to the T cells (e.g. via signaling from a costimulatory receptor). In some embodiments, the selection agent and at least one of the one or more stimulating agents are different. In some embodiments, the selection agent and each of the one or more stimulating agents are different. In some embodiments, an agent may be used both as a selection agent and as one of the one or more stimulating agent in connection with the provided methods. In some embodiments, the one or more stimulatory agent are bound on a reagent that delivers the stimulatory signal to the cells (e.g. stimulatory reagent). In some embodiments, the reagent contains a plurality of binding sites for binding each of the one or more stimulatory agent such that the stimulatory agents are multimerized on the agent. In particular embodiments, such a stimulatory reagent is an oligomeric or polymeric reagent made up of multiple individual molecules, such as multiple protein units or complexes (e.g. tetramers). Exemplary stimulatory reagents to which the one or more stimulatory agents are bound, including oligomeric stimulatory reagents, for use in connection with the provided devices and methods are described in Secion II.B.2. In particular embodiments, the stimulatory reagent is added to the chromatography column containing the immobilized cells under conditions suitable for delivering a signal in the cells. For instance, the on-column stimulation is carried out at appropriate temperatures as described herein by heating the device as described and provided herein to a physiologic temperature appropriate to permit cellular signaling events in the cells, such as a temperate of at or about between 30 °C and at or about 39 °C, for example at or about 37 °C ± 2 °C, such as at or about 37 °C.

[0316] In some embodiments, the stimulatory reagent to which the one or more stimulatory agent are bound provides a reversible system in which the one or more stimulatory agent are reversibly associated with the ewagent. Exemplary reversible systems for stimulation of cells include those described in WO2015 / 158868, WO2017068421, or WO2018 / 197949. In some embodiments, the reversible system employs a reagent composed of oligomers or polymers of a streptavidin mutein that reversibly bind to the one or more stimulatory agent via a streptavidin-binding peptide binding partner contained by the one or more stimulatory agent. In some embodiments, adding a free binding partner or competition agent (also called competition substance) disrupts the binding between the one or more stimulatory agent and the reagent, thereby reversing binding of the one or more stimulatory agent from the reagent and terminating or disrupting the stimulatory signal delivered by the one or more stimulatory agents of the stimulatory reagent. For instance, in the case of a streptavidin mutein / streptavidin binding peptide system an exemplary compeitition agent is biotin or a biotin analog (e.g. D-Biotin).

[0317] In particular aspects, using a device disclosed herein, provided herein are methods, which are not in accordance with the claimed invention, that employ reversible systems in which at least one agent (e.g., a selection agent or stimulatory agent) capable of binding to a molecule on the surface of a cell (cell surface molecule), is reversibly associated with a reagent (e.g., selection reagent or stimulatory reagent). In some cases, the reagent contains a plurality of binding sites capable of reversibly binding to the agent (e.g., a selection agent or stimulatory agent). In some cases, the reagent (e.g., selection reagent or stimulatory reagent) is a multimerization reagent. In some embodiments, the at least one agent (e.g., a selection agent or stimulatory agent) contains at least one binding site B that can specifically bind an epitope or region of the molecule and also contains a binding partner C that specifically binds to at least one binding site Z of the reagent (e.g., selection reagent or stimulatory reagent). In some cases, the binding interaction between the binding partner C and the at least one binding site Z is a non-covalent interaction. In some embodiments, the binding interaction, such as non-covalent interaction, between the binding partner C and the at least one binding site Z is reversible.

[0318] In some embodiments, the reversible association can be mediated in the presence of a substance, such as a competition agent or free binding agent, that is or contains a binding site that also is able to bind to the at least one binding site Z. Generally, the substance (e.g. competition agent or free binding agent) can act as a competitor due to a higher binding affinity for the binding site Z present in the reagent and / or due to being present at higher concentrations than the binding partner C, thereby detaching and / or dissociating the binding partner C from the reagent. In some embodiments, the affinity of the substance (e.g. competition agent or free binding agent) for the at least one binding site Z is greater than the affinity of the binding partner C of the agent (e.g., a selection agent or stimulatory agent) for the at least one binding site Z. Thus, in some cases, the bond between the binding site Z of the reagent and the binding partner C of the agent (e.g., a selection agent or stimulatory agent) can be disrupted by addition of the substance (e.g. competition agent or free binding partner), thereby rendering the association of the agent (e.g., a selection agent or stimulatory agent) and reagent (e.g., selection reagent or stimulatory reagent) reversible.

[0319] Reagents that can be used in such reversible systems are described and known in the art, see e.g., U.S. Patent Nos. 5,168,049; 5,506,121; 6,103,493; 7,776,562; 7,981,632; 8,298,782; 8,735,540; 9,023,604; and International published PCT Appl. Nos. WO2013 / 124474 and WO2014 / 076277. Non-limiting examples of reagents and binding partners capable of forming a reversible interaction, as well as substances (e.g. competition agents or free binding agents) capable of reversing such binding, are described below.1. Agents

[0320] In some embodiments, the agent (e.g., selection agent or stimulatory agent) has one or more binding sites, B, for binding to the molecule on the surface of the cell, e.g. cell surface molecule. Thus, in some instances, the agent (e.g., selection agent or stimulatory agent) contains a binding site B or a plurality of binding sites B, wherein the specific binding between the agent (e.g., selection agent or stimulatory agent) and the molecule on the surface of the target cells contains interaction between B and the molecule. In some embodiments, the agent contains only a single binding site, i.e. is monovalent. In some embodiments, the agent (e.g., selection agent or stimulatory agent) has at least two, such as a plurality of binding sites B including three, four or five binding sites B capable of binding to the cell surface molecule. In some such aspects, the at least two or plurality of binding sites B may be identical. In some embodiments, one or more of the at least two or plurality of binding sites B may be different (e.g. B1 and B2).

[0321] In some embodiments, one or more different agents (e.g. one or more different e.g., selection agent or stimulatory agent or other agent that binds to a molecule on a cell) are reversibly bound to the reagent (e.g., selection reagent or stimulatory reagent). In some embodiments, at least 2, 3, 4 or more different agents (e.g., selection agents or stimulatory agents) are reversibly bound to the same reagent. In some embodiments, at least two different agents (e.g., selection agent or stimulatory agents) are reversibly bound to the same reagent, whereby each agent comprises a binding site B or a plurality of binding sites B for specific binding between the agent and the molecule. In some embodiments, the at least two or more agents (e.g., selection agent or stimulatory agents) contain the same binding site B, e.g. for the binding the same or substantially the same molecule. In some embodiments, the at least two or more agents (e.g., selection agents or stimulatory agents) contain different binding sites B, e.g. for the binding to different molecules. In some embodiments, a first agent (e.g., a first selection agent or first stimulatory agent) contains a binding site B1, B2, B3, B4, etc. and a second agent (e.g., second selection agent or second stimulatory agent) contains another of a binding site B1, B2, B3, B4, etc. In some embodiments, a first agent (e.g. a first selection agent) contains a binding site B1 and a second agent (e.g. second selection agent) contains a binding site B3. In some embodiments, a first agent (e.g. a first stimulatory agent) contains a binding site B2 and a second agent (e.g. a second stimulatory agent) contains a binding site B4. In any of such embodiments, the first agent and second agent can contain a binding partner, C1 or C2. In some embodiments, C1 and C2 can be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and second agent contain the same binding partner, C1.

[0322] In some cases, the dissociation constant (K D ) of the binding between the agent (e.g., via the binding site B) and the binding site Z of the reagent may have a value in the range from about 10 -2< M to about 10 -13< M or from about 10 -3< M to about 10 -12< M or from about 10 -4< M to about 10 -11< M, or from about 10 -5< M to about 10 -10< M. In some embodiments, the dissociation constant (K D ) for the binding between the binding agent and the molecule is of low affinity, for example, in the range of a K D of about 10 -3< to about 10 -7< M. In some embodiments, the dissociation constant (K D ) for the binding between the binding agent and the molecule is of high affinity, for example, in the range of a K D of about 10 -7< to about 1×10 -10< M.

[0323] In some embodiments, the dissociation of the binding of the agent via the binding site B and the molecule occurs sufficiently fast, for example, to allow the target cell to be only transiently stained or associated with the agent after disruption of the reversible bond between the reagent and the agent. In some cases, when expressed in terms of the k off rate (also called dissociation rate constant for the binding between the agent (via the binding site B) and the molecule, the k off rate is about 0.5×10 -4< sec -1< or greater, about 1×10 -4< sec -1< or greater, about 2×10 -4< sec -1< or greater, about 3 × 10 -4< sec -1< or greater, about 4×10 -4< sec -1< of greater, about 5×10 -4< sec -1< or greater, about 1×10 -3< sec -1< or greater, about 1.5×10 -3< sec -1< or greater, about 2×10 -3< sec -1< or greater, about 3×10 -3< sec -1< or greater, about 4×10 -3< sec -1< , about 5×10 -3< sec -1< or greater, about 1×10 -2< sec or greater, or about 5×10 -1< sec -1< or greater. It is within the level of a skilled artisan to empirically determine the k off rate range suitable for a particular agent and cell molecule interaction (see e.g. U.S. published application No. US2014 / 0295458). For example, an agent with a rather high k off rate of, for example, greater than 4.0×10 -4< sec -1< may be used so that, after the disruption of the binding complexes, most of the agent can be removed or dissociated within one hour. In other cases, an agent with a lower k off rate of, for example, 1.0×10 -4< sec -1< , may be used, so that after the disruption of the binding complexes, most of the agent may be removed or dissociated from the cell within about 3 and a half hours.

[0324] In some embodiments, the K D of this bond as well as the K D , k off and k on rate of the bond formed between the binding site B of the agent (e.g., e.g., selection agent or stimulatory agent) and the cell surface molecule can be determined by any suitable means, for example, by fluorescence titration, equilibrium dialysis or surface plasmon resonance.

[0325] In some aspects, the cell surface molecule is a molecule against which an agent (e.g., selection agent or stimulatory agent) may be directed. In some embodiments, the cell surface molecule is a peptide or a protein, such as a receptor, e.g., a membrane receptor protein. In some embodiments, the receptor is a lipid, a polysaccharide or a nucleic acid. In some embodiments, a cell surface molecule that is a protein may be a peripheral membrane protein or an integral membrane protein. The cell surface molecule may in some embodiments have one or more domains that span the membrane. As a few illustrative examples, a membrane protein with a transmembrane domain may be a G-protein coupled receptor, such as an odorant receptors, a rhodopsin receptor, a rhodopsin pheromone receptor, a peptide hormone receptor, a taste receptor, a GABA receptor, an opiate receptor, a serotonin receptor, a Ca2+ receptor, melanopsin, a neurotransmitter receptor, such as a ligand gated, a voltage gated or a mechanically gated receptor, including the acetylcholine, the nicotinic, the adrenergic, the norepinephrine, the catecholamines, the L-DOPA-, a dopamine and serotonin (biogenic amine, endorphin / enkephalin) neuropeptide receptor, a receptor kinase such as serine / threonine kinase, a tyrosine kinase, a porin / channel such as a chloride channel, a potassium channel, a sodium channel, an OMP protein, an ABC transporter (ATP-Binding Cassette-Transporter) such as amino acid transporter, the Na-glucose transporter, the Na / iodide transporter, an ion transporter such as Light Harvesting Complex, cytochrome c oxidase, ATPase Na / K, H / K, Ca, a cell adhesion receptor such as metalloprotease, an integrin or a catherin.

[0326] In some embodiments, the cell surface molecule may be an antigen defining a desired cell population or subpopulation, for instance a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, T-helper cells, for example, CD4+ T-helper cells, B cells or natural killer cells), monocytes, or stem cells, e.g. CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells. Examples of T-cells include cells such as CMV-specific CD8+ T-lymphocytes, cytotoxic T-cells, memory T-cells and regulatory T-cells (Treg). An illustrative example of Treg is CD4 CD25 CD45RA Treg cells and an illustrative example of memory T-cells is CD62L CD8+ specific central memory T-cells. The cell surface molecule may also be a marker for a tumor cell.

[0327] As described above, in some embodiments, the agent (e.g., selection agent or stimulatory agent) has, in addition to the binding site B that is able to bind the cell surface molecule, a binding partner C. In some aspects, this binding partner C is able to bind to a binding site Z of the reagent (e.g., selection reagent or stimulatory reagent (e.g., oligomeric stimulatory reagent)) wherein the reagent has one or more binding sites for the binding partner C. In some embodiments, the non-covalent bond that may be formed between the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) and the binding site(s) Z of the reagent (e.g., selection reagent or stimulatory reagent (e.g., oligomeric stimulatory reagent)) may be of any desired strength and affinity, and may be disruptable or reversible under conditions under which the method is performed. The agent (e.g., receptor-binding agent or selection agent) may include at least one, including two, three or more, additional binding partners C and the reagent (e.g., selection reagent or stimulatory reagent (e.g., oligomeric stimulatory reagent)) may include at least two, such as three, four, five, six, seven, eight or more binding sites Z for the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent). As described in US patent 7,776,562, US patent 8,298,782 or International Patent application WO 2002 / 054065, any combination of a binding partner C and a reagent with one or more corresponding binding sites Z can be chosen, for example, such that the binding partner C and the binding site Z are able to reversibly bind in a complex, such as to cause an avidity effect.

[0328] The binding partner C included in the agent (e.g., selection agent or stimulatory agent) may for instance be hydrocarbon-based (including polymeric) and include nitrogen-, phosphorus-, sulphur-, carben-, halogen- or pseudohalogen groups. In some aspects, it may be an alcohol, an organic acid, an inorganic acid, an amine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a saccharide, an oligosaccharide, or a polysaccharide. As further examples, it may also be a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube or carbon nanofoam. Generally, such a binding partner C has a higher affinity to the binding site of the reagent than to other matter. Examples of a respective binding partner C include, but are not limited to, a crown ether, an immunoglobulin, a fragment thereof and a proteinaceous binding molecule with antibody-like functions.

[0329] In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes biotin and the reagent includes a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes a biotin analog that reversibly binds to streptavidin or avidin, and the reagent includes streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes a streptavidin or avidin binding peptide and the reagent includes streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective streptavidin or avidin binding peptide. For purposes herein, the term analog is used interchangeably with the term mutein in reference to a mutant form of a streptavidin (e.g. streptavidin analog or streptavidin mutein) or an avidin (e.g. avidin analog or avidin mutein).

[0330] In some embodiments, the reagent (e.g., selection reagent or stimulatory reagent) is or contains a streptavidin, such as a streptavidin mutein including any described above (e.g. set forth in SEQ ID NOS: 3-6), and the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) may include a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide may include a sequence with the general formula set forth in SEQ ID NO: 9, such as contains the sequence set forth in SEQ ID NO: 10. In some embodiments, the streptavidin-binding peptide sequence has the general formula set forth in SEQ ID NO: 11, such as set forth in SEQ ID NO: 12. In one example, the streptavidin-binding peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag ®< , set forth in SEQ ID NO: 7). In one example, the streptavidin-binding peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag ®< II, set forth in SEQ ID NO: 8). In some embodiments, the streptavidin-binding peptide ligand contains a sequential arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is at least 0 and not greater than 50 amino acids, wherein one binding module has 3 to 8 amino acids and contains at least the sequence His-Pro-Xaa (SEQ ID NO: 9), where Xaa is glutamine, asparagine, or methionine, and wherein the other binding module has the same or different streptavidin peptide ligand, such as set forth in SEQ ID NO: 11 (see e.g. International Published PCT Appl. No. WO02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the streptavidin-binding peptide ligand contains a sequence having the formula set forth in any of SEQ ID NO: 13 or 14. In some embodiments, the streptavidin-binding peptide ligand has the sequence of amino acids set forth in any of SEQ ID NOS: 15-19. In most cases, all these streptavidin binding peptides bind to the same binding site, namely the biotin binding site of streptavidin. If one or more of such streptavidin binding peptides is used as binding partners C, e.g. C1 and C2, the multimerization reagent is typically a streptavidin mutein.

[0331] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide may include the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag ®< II, set forth in SEQ ID NO: 8) conjugated with a nickel charged trisNTA (also called His-STREPPER or His / Strep-tag ®< II Adapter).

[0332] In some embodiments, the binding partner C of the agent (e.g., receptor-binding agent or selection agent) includes a moiety known to the skilled artisan as an affinity tag. In such an embodiment, the reagent may include a corresponding binding partner, for example, an antibody or an antibody fragment, known to bind to the affinity tag. As a few illustrative examples of known affinity tags, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) may include dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), chitin binding protein (CBP) or thioredoxin, calmodulin binding peptide (CBP), FLAG '-peptide, the HA-tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (SEQ ID NO: 20), the VSV-G-tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys) (SEQ ID NO: 21), the HSV-tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp) (SEQ ID NO: 22), the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly) (SEQ ID NO: 23), maltose binding protein (MBP), the HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 24) of herpes simplex virus glycoprotein D, the "myc" epitope of the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 25), the V5-tag (sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) (SEQ ID NO: 26), or glutathione-S-transferase (GST). In such embodiments, the complex formed between the one or more binding sites Z of the reagent which may be an antibody or antibody fragment, and the antigen can be disrupted competitively by adding the free antigen, i.e. the free peptide (epitope tag) or the free protein (such as MBP or CBP). In some embodiments, the affinity tag might also be an oligonucleotide tag. In some cases, such an oligonucleotide tag may, for instance, be used to hybridize to an oligonucleotide with a complementary sequence, linked to or included in the reagent.

[0333] Further examples of a suitable binding partner C include, but are not limited to, a lectin, protein A, protein G, a metal, a metal ion, nitrilo triacetic acid derivatives (NT A), RGD-motifs, a dextrane, polyethyleneimine (PEI), a redox polymer, a glycoproteins, an aptamers, a dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatine, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, poly U, or oligo-dT. Lectins such as Concavalin A are known to bind to polysaccharides and glycosylated proteins. An illustrative example of a dye is a triazine dye such as Cibacron blue F3G-A (CB) or Red HE-3B, which specifically bind NADH-dependent enzymes. Typically, Green A binds to Co A proteins, human serum albumin, and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. Generally, cations of metals such as Ni, Cd, Zn, Co, or Cu, are typically used to bind affinity tags such as an oligohistidine containing sequence, including the hexahistidine or the His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tag (MAT tag) (SEQ ID NO: 35), and N-methacryloyl-(L)-cysteine methyl ester.

[0334] In some embodiments, the binding between the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) and the one or more binding sites Z of the reagent occurs in the presence of a divalent, a trivalent or a tetravalent cation. In this regard, in some embodiments, the reagent includes a divalent, a trivalent or a tetravalent cation, typically held, e.g. complexed, by means of a suitable chelator. In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) may include a moiety that includes, e.g. complexes, a divalent, a trivalent or a tetravalent cation. Examples of a respective metal chelator, include, but are not limited to, ethylenediamine, ethylene-diaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetri-aminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimer-capto-1-propanol (dimercaprol), porphine and heme. As an example, EDTA forms a complex with most monovalent, divalent, trivalent and tetravalent metal ions, such as e.g. silver (Ag +< ), calcium (Ca 2+< ), manganese (Mn 2+< ), copper (Cu 2+< ), iron (Fe 2+< ), cobalt (Co +< ) and zirconium (Zr 4+< ), while BAPTA is specific for Ca 2+< . As an illustrative example, a standard method used in the art is the formation of a complex between an oligohistidine tag and copper (Cu 2+< ), nickel (Ni 2+< ), cobalt (Co 2+< ), or zinc (Zn 2+< ) ions, which are presented by means of the chelator nitrilotriacetic acid (NTA).

[0335] In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes a calmodulin binding peptide and the reagent includes multimeric calmodulin as described in US Patent 5,985,658, for example. In some embodiments, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes a FLAG peptide and the reagent includes an antibody that binds to the FLAG peptide, e.g. the FLAG peptide, which binds to the monoclonal antibody 4E11 as described in US Patent 4,851,341. In one embodiment, the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) includes an oligohistidine tag and the reagent includes an antibody or a transition metal ion binding the oligohistidine tag. In some cases, the disruption of all these binding complexes may be accomplished by metal ion chelation, e.g. calcium chelation, for instance by adding EDTA or EGTA. In some embodiments, calmodulin, antibodies such as 4E11 or chelated metal ions or free chelators may be multimerized by conventional methods, e.g. by biotinylation and complexation with streptavidin or avidin or oligomers thereof or by the introduction of carboxyl residues into a polysaccharide, e.g. dextran, essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137 in a first step and linking calmodulin or antibodies or chelated metal ions or free chelators via primary amino groups to the carboxyl groups in the polysaccharide, e.g. dextran, backbone using conventional carbodiimide chemistry in a second step. In some such embodiments, the binding between the binding partner C that is included in the agent (e.g., selection agent or stimulatory agent) and the one or more binding sites Z of the reagent can be disrupted by metal ion chelation. The metal chelation may, for example, be accomplished by addition of EGTA or EDTA.

[0336] In some embodiments, the agent (e.g., selection agent or stimulatory agent), which specifically bind to the cell surface molecule, may for instance be comprised by an antibody, a fragment thereof, or a proteinaceous binding molecule with antibody-like functions. In some embodiments, the binding site B of the agent is an antibody combining site, such as is or contains one or more complementarity determining regions (CDRs) of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single-chain Fv fragments (scFv), a divalent antibody fragment such as an (Fab)2'-fragment, diabodies, triabodies (Iliades, P., et al, FEB S Lett (1997) 409, 437-441), decabodies (Stone, E., et al, Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J., et al, Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, the agent (e.g., receptor-binding agent or selection agent) may comprise a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein that is also known as "duocalin".

[0337] In some embodiments, the agent (e.g., selection agent or stimulatory agent) may have a single binding site B, i.e., it may be monovalent. Examples of monovalent agents (e.g., selection agent or stimulatory agent) include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.

[0338] In some embodiments, the agent (e.g., selection agent or stimulatory agent) is an antibody or an antigen-binding fragment thereof, such as a Fab fragments, Fv fragments, single-chain Fv fragments (scFv), a divalent antibody fragment such as an F(ab') 2 -fragment. In some embodiments, the agent (e.g., selection agent or stimulatory agent) is or is derived from a parental antibody that is known to bind to a cell molecule of interest. Various antibody molecules or fragments thereof against cell surface molecules are well known in the art and any of a variety of such can be used as agents in the methods herein. In some embodiments, the agent (e.g., selection agent or stimulatory agent) is an antibody or fragment thereof that contains one or more amino acid replacements in the variable heavy chain of a parental or reference antibody, for example, to generate an antibody with an altered affinity or that exhibits a sufficiently fast off-rate as described above. For example, exemplary of such mutations are known the context of mutants of the anti-CD4 antibody 13B8.2 (see e.g., U.S. Patent Nos. 7,482,000, U.S. Patent Appl. Pub. No. US2014 / 0295458 or International Patent Application App. No. WO2013 / 124474), and any of such mutations can be generated in another parental or reference antibody.

[0339] In some aspects, the agent (e.g., selection agent or stimulatory agent) that can be monovalent, for example comprise a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or other) such as a mutein based on a polypeptide of the lipocalin family (also known as "Anticalin ®< ), or a bivalent molecule such as an antibody or a fragment in which both binding sites are retained such as an F(ab') 2 fragment.

[0340] An example of a proteinaceous binding molecule with antibody-like functions includes a mutein based on a polypeptide of the lipocalin family (see for example, WO 03 / 029462, Beste et al, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903). Generally, lipocalins, such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apo lipoprotein D or human tear lipocalin possess natural ligand-binding sites that can be modified so that they bind a given target. Further examples of a proteinaceous binding molecule with antibody-like binding properties that can be used as agent (e.g., selection agent or stimulatory agent) that specifically binds to the cell surface molecule include, but are not limited to, the so-called glubodies (see e.g. international patent application WO 96 / 23879), proteins based on the ankyrin scaffold (Mosavi, L.K., et al, Protein Science (2004) 13, 6, 1435-1448) or crystalline scaffold (e.g. international patent application WO 01 / 04144) the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, AdNectins, tetranectins and avimers. Generally, avimers, including multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains, contain so called A-domains that occur as strings of multiple domains in several cell surface receptors (Silverman, J., et al, Nature Biotechnology (2005) 23, 1556-1561). Adnectins, generally derived from a domain of human fibronectin, typically contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins, generally derived from the respective human homotrimeric protein, likewise typically contain loop regions in a C-type lectin domain that can be engineered for desired binding. Peptoids, which can, in some cases, act as protein ligands, typically are oligo(N-alkyl) glycines that differ from peptides in that the side chain is connected to the amide nitrogen rather than the carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have a much higher cell permeability than peptides (see e.g. Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

[0341] Further examples of suitable proteinaceous binding molecules include, but are not limited to, an EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz / Bovine pancreatic trypsin Inhibitor domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or a an immunoglobulin-like domain (for example, domain antibodies or camel heavy chain antibodies), a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP -type four disulfide core domain, a F5 / 8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, "Kappabodies" (Ill et al. Protein Eng (1997) 10, 949-57, a so called "minibody" (Martin et al, EMBO J (1994) 13, 5303-5309), a diabody (Holliger et al, PNAS USA (1993)90, 6444-6448), a so called "Janusis" (Traunecker et al, EMBO J (1991) 10, 3655-3659, or Traunecker et al, Int J Cancer (1992) Suppl 7, 51-52), a nanobody, a microbody, an affilin, an affibody, a knottin, ubiquitin, a zinc-finger protein, an autofluorescent protein or a leucine-rich repeat protein. In some embodiments, a nucleic acid molecule with antibody-like functions can be an aptamer. Generally, an aptamer folds into a defined three-dimensional motif and shows high affinity for a given target structure.a. Selection Agents

[0342] In certain aspects, the methods provided herein employ a selection agent. In some embodiments, the agent, as described in Section II-B, is a selection agent. In some embodiments, the selection agent binds to a molecule on the surface of a cell, such as a cell surface molecule. In some instances, the cell surface molecule is a selection marker. In some embodiments, the selection agent is capable of specifically binding to a selection marker expressed by one or more of the cells in a sample. In some embodiments, reference to specific binding to a molecule, such as a cell surace molecule or cell surface receptor, throughout the disclosure does not necessarily mean that the agent binds only to such molecule. For example, an agent that specifically binds to a molecule may bind to other molecules, generally with much lower affinity as determined by, e.g., immunoassays, BIAcore ®< , KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays. In some cases, the ability of an agent, under specific binding conditions, to bind to a target molecule such that its affinity or avidity is at least 5 times as great, such as at least 10, 20, 30, 40, 50, 100, 250 or 500 times as great, or even at least 1000 times as great as the average affinity or avidity of the same agent to a collection of random peptides or polypeptides of sufficient statistical size.

[0343] In some embodiments, the cells, e.g., target cells (e.g., T cells), have or express a molecule on the cell surface, e.g., a selection marker, such that the cells to be selected are defined by the presence of at least one common specific molecule (e.g., selection marker). In some embodiments, the sample containing the target cell may also contain additional cells that are devoid of the molecule (e.g., selection marker). For example, in some embodiments, T cells may be selected from a sample containing multiple cells types, e.g., red blood cells or B cells. Selection marker and receptor molecule may be used interchangeably herein to refer to a cell surface molecule.

[0344] In some embodiments, the selection agent is or contains an agent selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof; and / or the selection agent contains an antibody fragment; the selection agent is or contains a Fab fragment; the selection agent is selected from the group of divalent antibody fragments consisting of F(ab) 2 '-fragments and divalent single-chain Fv (scFv) fragments; the selection agent is a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFvs; and / or the selection agent is a proteinaceous binding molecule with antibody-like binding properties, selected from the group consisting of aptamers, muteins based on a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins, and avimers.

[0345] In some embodiments, the selection agent further contains a binding partner C for binding to the reagent. In some embodiments, the selection agent further contains biotin, a biotin analog that reversibly binds to a streptavidin or avidin, a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO:16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody binding the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody binding the oligohistidine tag.

[0346] In some embodiments, the reagent is or contains a streptavidin, streptavidin mutein, aviding or avidin mutein, and the selection agent contains a binding partner C that is able to bind the such reagent, such as biotin, a biotin analog or a streptavidin-binding peptide. In some embodiments, the selection agent further comprises biotin, a biotin analog that reversibly binds to a streptavidin or avidin, a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO:16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19). In particular embodiments, the reagent is or contains a streptavidin mutein (e.g. set forth in SEQ ID NO:6) and the binding partner C is a streptavidin-binding peptide, such as any set forth in any one of SEQ ID NOS: 8 or 15-19. In some embodiments, the the selection agent further comprises a streptavidin-binding peptide having the sequence SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0347] In some aspects, the cell surface molecule, e.g., selection marker, may be an antigen defining a desired cell population or subpopulation, for instance a population or subpopulation of blood cells, e. g. lymphocytes (e.g. T cells, T-helper cells, for example, CD4+ T-helper cells, B cells or natural killer cells), monocytes, or stem cells, e.g. CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells. In some embodiments, the selection marker can be a marker expressed on the surface of T cells or a subset of T cells, such as CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO Examples of T-cells include cells such as CMV-specific CD8+ T-lymphocytes, cytotoxic T-cells, memory T-cells and regulatory T-cells (Treg). An illustrative example of Treg includes CD4 CD25 CD45RA Treg cells and an illustrative example of memory T-cells includes CD62L CD8+ specific central memory T-cells.

[0348] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD3+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more selection agents that specifically binds to such markers. The selection agent may be any binding molecule, such as an antibody or antibody fragment, that binds to such surface markers to effect the positive or negative selection of T cells or subpopulations thereof.

[0349] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive-like, memory, and / or effector T cell subpopulations.

[0350] In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al., (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0351] In embodiments, memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies as selection agents.

[0352] In some embodiments, the enrichment for central memory T (T CM ) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. In some embodiments, the selection for the CD4+ cell population and the selection for the CD8+ cell population are carried out simultaneously. In some embodiments, the CD4+ cell population and the selection for the CD8+ cell population are carried out sequentially, in either order. In some embodiments, methods for selecting cells can include those as described in published U.S. App. No. US20170037369.

[0353] In particular embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0354] In some embodiments, a selection agent that specifically binds CD4 and a selection agent that specifically binds CD8 are used to generate a population enriched in CD4+ T cells and a population enriched in CD8+ T cells, respectively.

[0355] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0356] CD4+ T helper cells may be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0357] In some embodiments, the selection marker is a T cell coreceptor; the selection marker is or contains a member of a T cell antigen receptor complex; the selection marker is or contains a CD3 chain; the selection marker is or contains a CD3 zeta chain; the selection marker is or contains a CD8; the selection marker is or contains a CD4; the selection marker is or contains CD45RA; the selection marker is or contains CD27; the selection marker is or contains CD28; and / or the selection marker is or contains CCR7. In some embodiments, the selection marker is selected from the group consisting of CD3, CD4, and CD8. In some embodiments, the selection marker is CD3.

[0358] In some embodiments, the specific binding between the selection agent and the selection marker does not induce a signal, or does not induce a stimulatory or activating or proliferative signal, to the T cells. In some embodiments, the selection agent includes a monovalent antibody fragment that binds to CD3, CD8 or CD4. In some embodiments, the selection agent is an anti-CD3 Fab, an anti-CD8 Fab or an anti-CD4 Fab. In some embodietns, the selection agent is an anti-CD3 Fab. In some embodiments, the anti-CD3 Fab comprises an OKT3 antibody Fab fragment. In some embodiments, the anti-CD3 Fab comprises a variable heavy chain having the sequence set forth by SEQ ID NO:31 and a variable light chain having the sequence set forth by SEQ ID NO:32.

[0359] In some embodiments, the selection marker may be CD4 and the selection agent specifically binds CD4. In some aspects, the selection agent that specifically binds CD4 may be selected from the group consisting of an anti-CD4-antibody, a divalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4-antibody, and a proteinaceous CD4 binding molecule with antibody-like binding properties. In some embodiments, an anti-CD4-antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g. CD4 Fab fragment) can be derived from antibody 13B8.2 or a functionally active mutant of 13B8.2 that retains specific binding for CD4. For example, exemplary mutants of antibody 13B8.2 or m13B8.2 are described in U.S. Patent Nos. 7,482,000, U.S. Patent Appl. No. US2014 / 0295458 or International Patent Application No. WO2013 / 124474; and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment termed "m13B8.2" carries the variable domain of the CD4 binding murine antibody 13B8.2 and a constant domain containing constant human CH1 domain of type gamma for the heavy chain and the constant human light chain domain of type kappa, as described in US Patent 7,482,000. In some embodiments, the anti-CD4 antibody, e.g. a mutant of antibody 13B8.2, contains the amino acid replacement H91A in the variable light chain, the amino acid replacement Y92A in the variable light chain, the amino acid replacement H35A in the variable heavy chain and / or the amino acid replacement R53A in the variable heavy chain, each by Kabat numbering. In some aspects, compared to variable domains of the 13B8.2 Fab fragment in ml3B8.2 the His residue at position 91 of the light chain (position 93 in SEQ ID NO: 30) is mutated to Ala and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO: 29) is mutated to Ala. In some embodiments, the reagent that is reversibly bound to anti-CD4 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g. c...

Claims

1. A chromatography column, comprising a housing assembly (1) for column chromatography, the housing assembly comprising: an inlet housing member (2) and an outlet housing member (3), wherein at least the inlet housing member and the outlet housing member form an internal cavity configured to house a stationary phase for column chromatography, wherein: the internal cavity comprises the stationary phase for column chromatography; and the stationary phase comprises an affinity chromatography matrix; a temperature control member configured to provide heat to the stationary phase in the internal cavity; and a connector (6) configured to operably connect the internal cavity to a gas source, thereby permitting or effecting intake of gas into the internal cavity.

2. The chromatography column of claim 1, the housing assembly further comprising a side wall member (7), wherein the inlet housing member, the outlet housing member, and the side wall member form the internal cavity.

3. The chromatography column of claim 1 or claim 2, wherein the connector comprises one or more filter(s), and optionally, wherein the one or more filter(s) is a gas filter.

4. The chromatography column of any one of claims 1-3, wherein the inlet housing member comprises one or more inlet(s) operably connected to the internal cavity to permit intake of an input composition into the internal cavity; and / or wherein the outlet housing member comprises one or more outlet(s) operably connected to the internal cavity to permit or effect discharge of an output composition from the internal cavity.

5. The chromatography column of any of claims 1-4, further comprising: a first porous member (9) configured to separate the stationary phase and an inlet of the internal cavity, wherein the first porous member is optionally between the inlet housing member and the side wall member, and further optionally, wherein the first porous member is independently a cell strainer or a cell sieve; and / or a second porous member (9) configured to separate the stationary phase and an outlet of the internal cavity, wherein the second porous member is optionally between the outlet housing member and the side wall member, and further optionally, wherein the second porous member is independently a cell strainer or a cell sieve.

6. The chromatography column of any one of claims 1-5, wherein the temperature control member is configured: to heat the stationary phase to a target temperature between about 30°C and about 39°C, and optionally, wherein the target temperature is between about 35°C and about 39°C, and further optionally at or about 37°C, and / or to maintain the stationary phase at the target temperature.

7. The chromatography column of any one of claims 1-6, wherein the temperature control member comprises one or more heating element(s), and optionally, wherein the one or more heating element(s) are configured to uniformly heat the stationary phase.

8. The chromatography column of claim 7, wherein at least one of the one or more heating element(s) is: a) an electromagnetic induction heating element, and the electromagnetic induction heating element comprises an induction heating coil surrounding a magnetizable core configured to provide heat to the stationary phase in the internal cavity; or b) an electric heating element (17) and optionally, wherein the electric heating element comprises a metal plate, a metal rod, a metal wire, or a combination thereof; and / or wherein the electric heating element is configured to connect to a power source external to the housing assembly; or c) a non-electric heating element, and the non-electric heating element comprises a heating channel comprising an inlet and an outlet for a heated fluid, and optionally, wherein the heating channel is a heating coil and / or wherein the heated fluid is heated water, and further optionally, wherein the inlet for heated water is configured to connect to an external reservoir of heated water.

9. The chromatography column of any one of claims 7-8, wherein at least one of the one or more heating element(s) is disposed along and / or around a central axis of the internal cavity, and / or wherein at least one of the one or more heating element(s) is disposed inside the internal cavity, outside the internal cavity, or partially inside and partially outside the internal cavity.

10. The chromatography column of any one of claims 7-9, wherein at least one of the one or more heating element(s) 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.

11. The chromatography column of any one of claims 7-10 when each is dependent on claim 2, wherein at least a portion of at least one of the one or more heating element(s) 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.

12. The chromatography column of any one of claims 7-10 when each is dependent on claim 1, wherein at least a portion of at least one of the one or more heating element(s) 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 a side wall member, optionally at least a portion of the side wall member.

13. The chromatography column of any one of claim 10 or of claims 11 or 12 when dependent on claim 10, wherein the housing assembly further comprises an insulation layer between at least one of the one or more heating element(s) 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, and optionally, wherein the insulation layer comprises a gas, optionally air, or a liquid.

14. The chromatography column of any one of claims 7-13, wherein the housing assembly further comprises a jacket member (12) comprising at least one of the one or more heating element(s), wherein 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 side wall member, and optionally: wherein the jacket member is releasably connected together to surround the at least a portion of the inlet housing member, the at least a portion of the outlet housing member, and / or the at least a portion of the side wall member; and / or wherein the jacket member is configured to surround at least a portion of the side wall member, optionally is configured to entirely surround the side wall member.

15. The chromatography column of any preceding claim, wherein the stationary phase comprises or is a non-magnetic material, a non-ferromagnetic material, or non-paramagnetic material.

16. The chromatography column of any preceding claim, wherein the stationary phase is configured to immobilize target cells thereon, optionally wherein the target cells are T cells, optionally CD3+, CD4+, or CD8+ T cells.

17. The chromatography column of any preceding claim, wherein the stationary phase comprises a selection agent (32) immobilized thereon.

18. The chromatography column of claim 17, wherein the selection agent is capable of specific binding to a selection marker (34) on the surface of one or more cell(s), and optionally wherein the one or more cell(s) are immune cells, further optionally T cells.

19. The chromatography column of claim 18, wherein the selection agent is or comprises an agent selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof; and / or the selection agent comprises an antibody fragment; the selection agent is or comprises a Fab fragment; the selection agent is or comprises a single domain antibody, optionally a VHH antibody; the selection agent is selected from the group of divalent antibody fragments consisting of F(ab')2 fragments and divalent single-chain Fv (scFv) fragments; the selection agent is a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments, and scFvs; and / or the selection agent is a proteinaceous binding molecule with antibody-like binding properties, selected from the group consisting of aptamers, muteins based on a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins, and avimers.

20. The chromatography column of one of claims 18 or 19, wherein: the selection marker is a T cell coreceptor; the selection marker is or comprises a member of a T cell antigen receptor complex; the selection marker is or comprises a CD3 complex; the selection marker is or comprises a CD3 chain; the selection 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.

21. The chromatography column of any one of claims 18-20, wherein the selection agent comprises or is an anti-CD3 Fab, an anti-CD8 Fab, an anti-CD4 Fab, or an anti-CD27 Fab.

22. The chromatography column of any one of claims 18-21, wherein the selection agent is bound indirectly to the stationary phase through a selection reagent (31) to which the selection agent reversibly binds.

23. The chromatography column of claim 22, wherein the selection reagent comprises or is a mutein of streptavidin that reversibly binds a streptavidin-binding peptide and optionally, wherein the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19); and / or, wherein the streptavidin mutein comprises the amino acid sequence Val44-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 of SEQ ID NO: 1, or the streptavidin mutein comprises the amino acid sequence Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 of SEQ ID NO: 1; and / or wherein the N-terminal amino acid residue of the streptavidin mutein is in 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 in the region of amino acids 133 to 142 of SEQ ID NO: 1, and / or wherein the streptavidin mutein comprises the amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27, 28, 104, and 105.

24. The chromatography column of any of claims 17-23, wherein the selection agent comprises a streptavidin-binding peptide, optionally wherein the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).