Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63/304,545, filed January 28, 2022, the contents of which are hereby incorporated by reference in their entirety for all purposes.

Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042025940SeqList.xml, created January 23, 2023, which is 58,946 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

Field

[0003] The present disclosure relates in some aspects to chromatography arrays for the isolation of cells. In some embodiments, the cells are isolated based on their expression or lack of expression of a selection marker, for instance a cell surface marker. Also provided herein are kits and methods for preparing the provided chromatography arrays, as well as methods for isolating target cells, including using any of the provided chromatography arrays.

Background

[0004] Various methods for the chromatographic isolation of cells are available. However, in some cases, some existing processes may result in an isolated cell population with reduced yield, viability, purity, or functional activity. In some cases, some existing processes are not suitable for automation or semi-automation, may be slow to perform, or are not compatible with high-throughput cell isolation methods. Improved devices and methods are needed.

Summary

[0005] Provided herein in some embodiments is a chromatography array comprising a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening, wherein the cavity of each of the one or more wells comprises a stationary phase, the stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell.

[0006] In some of any embodiments, the selection agent is reversibly immobilized to the chromatography matrix.

[0007] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing; and the selection agent comprises a binding partner that is bound to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

[0008] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin or a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin or streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin or streptavidin mutein.

[0009] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin mutein.

[0010] In some of any embodiments, the binding partner is reversibly bound to the protein reagent, thereby effecting the reversible immobilization of the selection agent, wherein the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some of any embodiments, the competition reagent comprises biotin or a biotin analog. In some of any embodiments, the competition reagent comprises biotin.

[0011] In some of any embodiments, the binding partner comprises biotin, a biotin analog, or a streptavidin-binding peptide. In some of any embodiments, the binding partner comprises a streptavidin-binding peptide. In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) or Trp- Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, the streptavidin- binding peptide comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0012] In some of any embodiments, the streptavidin-binding peptide comprises two or more streptavidin-binding modules. In some of any embodiments, each of the two or more streptavidin-binding modules comprises an amino acid sequence independently selected from Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, each of the two or more streptavidin-binding modules comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0013] In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15), SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu- Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu- Lys (SEQ ID NO: 18), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-A la-Trp-Ser-His-Pro-Gln-Phe- Glu-Lys (SEQ ID NO: 19). In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16).

[0014] In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to wild-type streptavidin set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to a minimal streptavidin sequence, wherein the minimal streptavidin sequence is a fragment of wild-type streptavidin that is shortened at the N- and/or the C-terminus. In some of any embodiments, the N-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 10 to 16 of the sequence of amino acids set forth in SEQ ID NO: 1, and the C-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 133 to 142 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an amino acid sequence from position 13 to position 139 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an N-terminal methionine residue instead of Alai 3. In some of any embodiments, the minimal streptavidin sequence comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0015] In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 41) or Val^-Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 42) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ilc^-Gly^-Ala^’-Arg ⁴⁷ (SEQ ID NO: 41) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, 28, 39, and 40. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6.

[0016] In some of any embodiments, the selection agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an MHC molecules, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing, that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody or an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a monovalent antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a Fab that specifically binds to the selection marker.

[0017] In some of any embodiments, the selection marker is a marker expressed on the surface of immune cells. In some of any embodiments, the selection marker is a marker expressed on the surface of lymphocytes. In some of any embodiments, the selection marker is a marker expressed on the surface of T cells.

[0018] In some of any embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, or CD45RO. In some of any embodiments, the selection marker is CD3.

[0019] In some of any embodiments, the chromatography matrix includes a non-magnetic or non-magnetizable material. [0020] In some of any embodiments, the chromatography matrix comprises a chromatography resin. In some of any embodiments, the chromatography matrix comprises a polystyrene resin. In some of any embodiments, the chromatography matrix comprises chromatography resin beads. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm, 75 pm and 125 pm, or 90 pm and 110 pm in diameter, each inclusive. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 75 pm and 125 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 90 pm and 110 pm in diameter, inclusive.

[0021] In some of any embodiments, the amount of stationary phase comprised in each cavity is capable of immobilizing between or between about 0.1 x 10 ⁶ and 20 x 10 ⁶ cells expressing the selection marker. In some of any embodiments, the amount of stationary phase comprised in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 20 x 10 ⁶ cells expressing the selection marker.

[0022] In some of any embodiments, the amount of stationary phase comprised in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker.

[0023] In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 10000 mg, inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 4000 mg, inclusive.

[0024] In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 3000 mg, 10 mg and 1000 mg, 100 mg and 500 mg, or 200 mg and 400 mg, each inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 3000 mg, inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 1000 mg, inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 100 mg and 500 mg, inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 200 mg and 400 mg, inclusive. [0025] In some of any embodiments, the barrier spans the bottom opening.

[0026] In some of any embodiments, the barrier is positioned at, flush with, and/or secured to the bottom opening. In some of any embodiments, the barrier is positioned at the bottom opening. In some of any embodiments, the barrier is flush with the bottom opening. In some of any embodiments, the barrier is secured to the bottom opening.

[0027] In some of any embodiments, the barrier is positioned and/or secured within the cavity. In some of any embodiments, the barrier is positioned within the cavity. In some of any embodiments, the barrier is secured within the cavity. In some of any embodiments, the barrier is secured to a sidewall of the cavity.

[0028] In some of any embodiments, the barrier is permeable to T cells. In some of any embodiments, the barrier is not permeable to the stationary phase. In some of any embodiments, the barrier comprises pores that are at least or at least about 10 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm, 15 pm and 100 pm, or 20 pm and 50 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 15 pm and 100 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 20 pm and 50 pm in diameter.

[0029] In some of any embodiments, the barrier comprises pores that are between or between about 30 pm and 40 pm in diameter.

[0030] In some of any embodiments, the barrier comprises polypropylene and/or polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media.

[0031] In some of any embodiments, the barrier comprises polypropylene media. In some of any embodiments, the barrier comprises polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media.

[0032] In some of any embodiments, the spatial array comprises, comprises about, or comprises at least 6 wells.

[0033] In some of any embodiments, the spatial array comprises, comprises about, or comprises at least 12 wells, 24 wells, 48 wells, or 96 wells. In some of any embodiments, the spatial array comprises at least 12 wells. In some of any embodiments, the spatial array comprises 24 wells. In some of any embodiments, the spatial array comprises 96 wells.

[0034] In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, 0.2 mL and 10 mL, or 0.5 mL and 1.5 mL, each inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 10 mL, inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.5 mL and 1.5 mL, inclusive.

[0035] In some of any embodiments, the spatial array comprises a multi-well plate. In some of any embodiments, the multi-well plate is a multi-well filter plate.

[0036] In some of any embodiments, the chromatography array is sterile. In some of any embodiments, the spatial array has been autoclaved. In some of any embodiments, the multiwell plate has been autoclaved. In some of any embodiments, the multi-well plate is made of material that can be sterilized. In some embodiments, the sterilizing comprises autoclaving the multi- well plate.

[0037] Also provided herein in some embodiments is a method for preparing a chromatography array, comprising adding a stationary phase to a cavity of each of one or more wells of a spatial array of wells, wherein each of the one or more wells comprises (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; and the stationary phase comprises a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell.

[0038] Also provided herein in some embodiments is a method for preparing a chromatography array, comprising (a) obtaining a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; and (b) adding a stationary phase to the cavity of each of the one or more wells, the stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell. [0039] In some of any embodiments, the method is for preparing the chromatography array of any of the provided embodiments.

[0040] In some of any embodiments, the method further comprises preparing the stationary phase prior to the adding, the preparing comprising immobilizing the selection agent on the chromatography matrix.

[0041] In some of any embodiments, the method further comprises centrifuging the chromatography array following the adding. In some of any embodiments, the steps of adding and centrifuging are repeated a plurality of times.

[0042] In some of any embodiments, the method further comprises sterilizing the spatial array. In some of any embodiments, the sterilizing comprises autoclaving the spatial array. In some of any embodiments, the spatial array comprises a multi-well plate. In some of any embodiments, the multi-well plate is made of material that can be sterilized. In some of any embodiments, the sterilizing comprises autoclaving the multi-well plate.

[0043] In some of any embodiments, the sterilizing is performed prior to the adding.

[0044] In some of any embodiments, the selection agent is reversibly immobilized to the chromatography matrix.

[0045] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing; and the selection agent comprises a binding partner that is bound to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

[0046] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin or a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin or streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin or streptavidin mutein.

[0047] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin mutein.

[0048] In some of any embodiments, the binding partner is reversibly bound to the protein reagent, thereby effecting the reversible immobilization of the selection agent, wherein the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some of any embodiments, the competition reagent comprises biotin or a biotin analog. In some of any embodiments, the competition reagent comprises biotin.

[0049] In some of any embodiments, the binding partner comprises biotin, a biotin analog, or a streptavidin-binding peptide. In some of any embodiments, the binding partner comprises a streptavidin-binding peptide. In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) or Trp- Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, the streptavidin- binding peptide comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0050] In some of any embodiments, the streptavidin-binding peptide comprises two or more streptavidin-binding modules. In some of any embodiments, each of the two or more streptavidin-binding modules comprises an amino acid sequence independently selected from Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, each of the two or more streptavidin-binding modules comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0051] In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15), SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu- Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu- Lys (SEQ ID NO: 18), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-A la-Trp-Ser-His-Pro-Gln-Phe- Glu-Lys (SEQ ID NO: 19). In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16). [0052] In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to wild-type streptavidin set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to a minimal streptavidin sequence, wherein the minimal streptavidin sequence is a fragment of wild-type streptavidin that is shortened at the N- and/or the C-terminus. In some of any embodiments, the N-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 10 to 16 of the sequence of amino acids set forth in SEQ ID NO: 1, and the C-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 133 to 142 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an amino acid sequence from position 13 to position 139 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an N-terminal methionine residue instead of Alai 3. In some of any embodiments, the minimal streptavidin sequence comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0053] In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 41) or Val^-Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 42) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ilc^-Gly^-Ala^’-Arg ⁴⁷ (SEQ ID NO: 41) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, 28, 39, and 40. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6.

[0054] In some of any embodiments, the selection agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an MHC molecules, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing, that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody or an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a monovalent antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a Fab that specifically binds to the selection marker.

[0055] In some of any embodiments, the selection marker is a marker expressed on the surface of immune cells. In some of any embodiments, the selection marker is a marker expressed on the surface of lymphocytes. In some of any embodiments, the selection marker is a marker expressed on the surface of T cells.

[0056] In some of any embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, or CD45RO. In some of any embodiments, the selection marker is CD3.

[0057] In some of any embodiments, the chromatography matrix includes a non-magnetic or non-magnetizable material.

[0058] In some of any embodiments, the chromatography matrix comprises a chromatography resin. In some of any embodiments, the chromatography matrix comprises a polystyrene resin. In some of any embodiments, the chromatography matrix comprises chromatography resin beads. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm, 75 pm and 125 pm, or 90 pm and 110 pm in diameter, each inclusive. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 75 pm and 125 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 90 pm and 110 pm in diameter, inclusive.

[0059] In some of any embodiments, the amount of stationary phase added to each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker.

[0060] In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 10000 mg, inclusive. In some of any embodiments, the amount of stationary phase comprised in each cavity is between or between about 10 mg and 4000 mg, inclusive.

[0061] In some of any embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 3000 mg, 10 mg and 1000 mg, 100 mg and 500 mg, or 200 mg and 400 mg, each inclusive. In some of any embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 3000 mg, inclusive. In some of any embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 1000 mg, inclusive. In some of any embodiments, the amount of stationary phase added to each cavity is between or between about 100 mg and 500 mg, inclusive. In some of any embodiments, the amount of stationary phase added to each cavity is between or between about 200 mg and 400 mg, inclusive.

[0062] In some of any embodiments, the barrier spans the bottom opening.

[0063] In some of any embodiments, the barrier is positioned at, flush with, and/or secured to the bottom opening. In some of any embodiments, the barrier is positioned at the bottom opening. In some of any embodiments, the barrier is flush with the bottom opening. In some of any embodiments, the barrier is secured to the bottom opening.

[0064] In some of any embodiments, the barrier is positioned and/or secured within the cavity. In some of any embodiments, the barrier is positioned within the cavity. In some of any embodiments, the barrier is secured within the cavity. In some of any embodiments, the barrier is secured to a sidewall of the cavity.

[0065] In some of any embodiments, the barrier is permeable to T cells. In some of any embodiments, the barrier is not permeable to the stationary phase. In some of any embodiments, the barrier comprises pores that are at least or at least about 10 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm, 15 pm and 100 pm, or 20 pm and 50 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 15 pm and 100 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 20 pm and 50 pm in diameter.

[0066] In some of any embodiments, the barrier comprises pores that are between or between about 30 pm and 40 pm in diameter.

[0067] In some of any embodiments, the barrier comprises polypropylene and/or polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media. [0068] In some of any embodiments, the barrier comprises polypropylene media. In some of any embodiments, the barrier comprises polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media.

[0069] In some of any embodiments, the spatial array comprises, comprises about, or comprises at least 12 wells, 24 wells, 48 wells, or 96 wells. In some of any embodiments, the spatial array comprises at least 12 wells. In some of any embodiments, the spatial array comprises 24 wells. In some of any embodiments, the spatial array comprises 96 wells.

[0070] In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, 0.2 mL and 10 mL, or 0.5 mL and 1.5 mL, each inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, each inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 10 mL, inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.5 mL and 1.5 mL, inclusive.

[0071] In some of any embodiments, the spatial array comprises a multi-well plate. In some of any embodiments, the multi-well plate is a multi-well filter plate.

[0072] In some of any embodiments, the chromatography array is sterile. In some of any embodiments, the spatial array has been autoclaved. In some of any embodiments, the multiwell plate has been autoclaved. In some of any embodiments, the multi-well plate is made of material that can be sterilized. In some embodiments, the sterilizing comprises autoclaving the multi- well plate.

[0073] Also provided herein in some embodiments is a kit for preparing a chromatography array, comprising (i) a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; (ii) a stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell; and (iii) instructions for preparing the chromatography array.

[0074] In some of any embodiments, the selection agent is reversibly immobilized to the chromatography matrix. [0075] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing; and the selection agent comprises a binding partner that is bound to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

[0076] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises streptavidin or a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin or streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin or streptavidin mutein.

[0077] In some of any embodiments, the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein the protein reagent is attached to the chromatography matrix and comprises a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin mutein. In some of any embodiments, the binding partner is bound to the biotin-binding site of the streptavidin mutein.

[0078] In some of any embodiments, the binding partner is reversibly bound to the protein reagent, thereby effecting the reversible immobilization of the selection agent, wherein the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some of any embodiments, the competition reagent comprises biotin or a biotin analog. In some of any embodiments, the competition reagent comprises biotin.

[0079] Also provided herein in some embodiments is a kit for preparing a chromatography array, comprising (i) a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; (ii) a chromatography matrix; (iii) a selection agent that is capable of being immobilized on the chromatography matrix and specifically binds to a selection marker expressed on the surface of a cell; and (iv) instructions for preparing the chromatography array. [0080] In some of any embodiments, the selection agent is capable of being reversibly immobilized to the chromatography matrix.

[0081] In some of any embodiments, a protein reagent is attached to the chromatography matrix and comprises streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing; and the selection agent comprises a binding partner that is capable of binding to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture. In some of any embodiments, the binding partner is capable of binding to the biotin-binding site of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

[0082] In some of any embodiments, a protein reagent is attached to the chromatography matrix and comprises streptavidin or a streptavidin mutein; and the selection agent comprises a binding partner that is capable of binding to the streptavidin or streptavidin mutein. In some of any embodiments, the binding partner is capable of binding to the biotin-binding site of the streptavidin or streptavidin mutein.

[0083] In some of any embodiments, a protein reagent is attached to the chromatography matrix and comprises a streptavidin mutein; and the selection agent comprises a binding partner that is capable of binding to the streptavidin mutein. In some of any embodiments, the binding partner is capable of binding to the biotin-binding site of the streptavidin mutein.

[0084] In some of any embodiments, the binding partner is capable of reversibly binding to the protein reagent to effect the reversible immobilization of the selection agent, wherein the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some of any embodiments, the competition reagent comprises biotin or a biotin analog. In some of any embodiments, the competition reagent comprises biotin.

[0085] In some of any of the provided embodiments, the kit is for preparing the chromatography array of any of the provided embodiments.

[0086] In some of any embodiments, the instructions are for performing any of the provided methods for preparing a chromatography array.

[0087] In some of any embodiments, the binding partner comprises biotin, a biotin analog, or a streptavidin-binding peptide. In some of any embodiments, the binding partner comprises a streptavidin-binding peptide. In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) or Trp- Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, the streptavidin- binding peptide comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0088] In some of any embodiments, the streptavidin-binding peptide comprises two or more streptavidin-binding modules. In some of any embodiments, each of the two or more streptavidin-binding modules comprises an amino acid sequence independently selected from Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8). In some of any embodiments, each of the two or more streptavidin-binding modules comprises the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

[0089] In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15), SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu- Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu- Lys (SEQ ID NO: 18), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-A la-Trp-Ser-His-Pro-Gln-Phe- Glu-Lys (SEQ ID NO: 19). In some of any embodiments, the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16).

[0090] In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to wild-type streptavidin set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises one or more mutations compared to a minimal streptavidin sequence, wherein the minimal streptavidin sequence is a fragment of wild-type streptavidin that is shortened at the N- and/or the C-terminus. In some of any embodiments, the N-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 10 to 16 of the sequence of amino acids set forth in SEQ ID NO: 1, and the C-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 133 to 142 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an amino acid sequence from position 13 to position 139 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the minimal streptavidin sequence comprises an N-terminal methionine residue instead of Alai 3. In some of any embodiments, the minimal streptavidin sequence comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0091] In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 41) or Val^-Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 42) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the amino acid sequence Ilc^-Gly^-Ala^’-Arg ⁴⁷ (SEQ ID NO: 41) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, 28, 39, and 40. In some of any embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6.

[0092] In some of any embodiments, the selection agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an MHC molecules, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing, that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody or an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises an antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a monovalent antibody fragment that specifically binds to the selection marker. In some of any embodiments, the selection agent comprises a Fab that specifically binds to the selection marker.

[0093] In some of any embodiments, the selection marker is a marker expressed on the surface of immune cells. In some of any embodiments, the selection marker is a marker expressed on the surface of lymphocytes. In some of any embodiments, the selection marker is a marker expressed on the surface of T cells.

[0094] In some of any embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, or CD45RO. In some of any embodiments, the selection marker is CD3.

[0095] In some of any embodiments, the chromatography matrix includes a non-magnetic or non-magnetizable material. [0096] In some of any embodiments, the chromatography matrix comprises a chromatography resin. In some of any embodiments, the chromatography matrix comprises a polystyrene resin. In some of any embodiments, the chromatography matrix comprises chromatography resin beads. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm, 75 pm and 125 pm, or 90 pm and 110 pm in diameter, each inclusive. In some of any embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 75 pm and 125 pm in diameter, inclusive. In some of any embodiments, the chromatography resin beads are between or between about 90 pm and 110 pm in diameter, inclusive.

[0097] In some of any embodiments, the barrier spans the bottom opening.

[0098] In some of any embodiments, the barrier is positioned at, flush with, and/or secured to the bottom opening. In some of any embodiments, the barrier is positioned at the bottom opening. In some of any embodiments, the barrier is flush with the bottom opening. In some of any embodiments, the barrier is secured to the bottom opening.

[0099] In some of any embodiments, the barrier is positioned and/or secured within the cavity. In some of any embodiments, the barrier is positioned within the cavity. In some of any embodiments, the barrier is secured within the cavity. In some of any embodiments, the barrier is secured to a sidewall of the cavity.

[0100] In some of any embodiments, the barrier is permeable to T cells. In some of any embodiments, the barrier is not permeable to the stationary phase. In some of any embodiments, the barrier comprises pores that are at least or at least about 10 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm, 15 pm and 100 pm, or 20 pm and 50 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 10 pm and 150 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 15 pm and 100 pm in diameter. In some of any embodiments, the barrier comprises pores that are between or between about 20 pm and 50 pm in diameter.

[0101] In some of any embodiments, the barrier comprises pores that are between or between about 30 pm and 40 pm in diameter. [0102] In some of any embodiments, the barrier comprises polypropylene and/or polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media.

[0103] In some of any embodiments, the barrier comprises polypropylene media. In some of any embodiments, the barrier comprises polyethylene media. In some of any embodiments, the barrier comprises polypropylene and polyethylene media.

[0104] In some of any embodiments, the spatial array comprises, comprises about, or comprises at least 12 wells, 24 wells, 48 wells, or 96 wells. In some of any embodiments, the spatial array comprises at least 12 wells. In some of any embodiments, the spatial array comprises 24 wells. In some of any embodiments, the spatial array comprises 96 wells.

[0105] In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, 0.2 mL and 10 mL, or 0.5 mL and 1.5 mL, each inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, each inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.2 mL and 10 mL, inclusive. In some of any embodiments, each of the one or more wells comprises a volume of between or between about 0.5 mL and 1.5 mL, inclusive.

[0106] In some of any embodiments, the spatial array comprises a multi-well plate. In some of any embodiments, the multi-well plate is a multi-well filter plate.

[0107] In some of any embodiments, the chromatography array is sterile. In some of any embodiments, the spatial array has been autoclaved. In some of any embodiments, the multiwell plate has been autoclaved. In some of any embodiments, the multi-well plate is made of material that can be sterilized. In some embodiments, the sterilizing comprises autoclaving the multi- well plate.

[0108] Also provided herein in some embodiments is a method for isolating a target cell, comprising (a) adding an input composition comprising a plurality of cells to the stationary phase of a well of the one or more wells of the chromatography array of any of the provided embodiments, the plurality of cells comprising one or more cells expressing the selection marker, thereby immobilizing the one or more cells on the stationary phase via binding of the selection marker to the selection agent; and (b) collecting a target cell from the chromatography array. [0109] Also provided herein in some embodiments is a method for isolating a target cell, comprising (a) obtaining the chromatography array of any of the provided embodiments; (b) adding an input composition comprising a plurality of cells to the stationary phase of a well of the one or more wells of the chromatography array, the plurality of cells comprising one or more cells expressing the selection marker, thereby immobilizing the one or more cells on the stationary phase via binding of the selection marker to the selection agent; and (c) collecting a target cell from the chromatography array.

[0110] In some of any embodiments, the target cell expresses the selection marker.

[0111] In some of any embodiments, the target cell expresses the selection marker, and the collecting comprises: binding of the selection agent to the selection marker expressed by the target cell; flow-through of non-target cells not expressing the selection marker through the barrier; and flow-through of the target cell through the barrier. In some of any embodiments, the collecting comprises adding biotin or a biotin analog to the stationary phase prior to the flowthrough of the target cell through the barrier. In some of any embodiments, the target cell expresses the selection marker, and the collecting comprises: binding of the selection agent to the selection marker expressed by the target cell; flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and flow-through of the target cell through the barrier.

[0112] In some of any embodiments, the collecting comprises adding biotin to the stationary phase prior to the flow-through of the target cell through the barrier. In some of any embodiments, the collecting comprises adding D-biotin to the stationary phase prior to the flowthrough of the target cell through the barrier. In some of any embodiments, the collecting comprises adding a biotin analog to the stationary phase prior to the flow-through of the target cell through the barrier. In some of any embodiments, the biotin analog is iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl- HABA.

[0113] In some of any embodiments, the target cell does not express the selection marker.

[0114] In some of any embodiments, the target cell does not express the selection marker, and the collecting comprises: binding of the selection agent to non-target cells expressing the selection marker; and flow-through of the target cell through the barrier. [0115] In some of any embodiments, the method comprises collecting an output composition of eluted cells that have passed through the barrier. In some of any embodiments, cells of the output composition comprise a lower proportion of cells that express the selection marker on their surface compared to the cells of the input composition.

[0116] Also provided herein in some embodiments is a method for isolating a target cell, comprising: (a) adding an input composition comprising at least 1 x 10 ⁶ cells/mL to each of one or more wells of a sterile multi-well plate, wherein one or more cells of the input composition express a selection marker, and wherein the multi- well plate comprises a plurality of wells, each well comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to one or more cells of the input composition and positioned proximal to the bottom opening, wherein the cavity of each of the one or more wells comprises a stationary phase, the stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to the selection marker so as to bind cells of the input composition that express the selection marker on their surface; (b) centrifuging the multi-well plate for 30 seconds to 10 minutes; and (c) collecting an output composition of eluted cells that have passed through the barrier, wherein cells of the output composition comprise a lower proportion of cells that express the selection marker on their surface compared to the cells of the input composition.

[0117] In some of any embodiments, the output composition of eluted cells comprises at least 30% of the cells of the input composition. In some of any embodiments, the output composition of eluted cells comprises 30% to 80% of the cells of the input composition.

[0118] In some of any embodiments, less than 30% of the cells of the output composition of eluted cells express the selection marker on their surface. In some of any embodiments, less than 20% of the cells of the output composition of eluted cells express the selection marker on their surface. In some of any embodiments, less than 10% of the cells of the output composition of eluted cells express the selection marker on their surface.

[0119] In some of any embodiments, the method further comprises expanding the cells of the output composition of eluted cells.

[0120] In some of any embodiments, the selection marker is a marker expressed on the surface of immune cells. In some of any embodiments, the selection marker is a marker expressed on the surface of lymphocytes. In some of any embodiments, the selection marker is a marker expressed on the surface of T cells.

[0121] In some of any embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, or CD45RO. In some of any embodiments, the selection marker is CD3.

[0122] In some of any embodiments, the target cell is an immune cell. In some of any embodiments, the target cell is a lymphocyte. In some of any embodiments, the target cell is a T cell.

[0123] In some of any embodiments, the input composition comprises between or between about 1 x 10 ⁶ cells and 50 x 10 ⁶ cells. In some of any embodiments, the input composition comprises between or between about 1 x 10 ⁶ cells and 40 x 10 ⁶ cells.

[0124] In some of any embodiments, the input composition comprises between or between about 10 x 10 ⁶ cells and 50 x 10 ⁶ cells.

[0125] In some of any embodiments, the input composition comprises at least 1 x 10 ⁶ cells/mL. In some of any embodiments, the input composition comprises at least 5 x 10 ⁶ cells/mL. In some of any embodiments, the input composition comprises at least 10 x 10 ⁶ cells/mL.

[0126] In some of any embodiments, the input composition comprises up to 50 x 10 ⁶ cells/mL.

[0127] In some of any embodiments, the input composition comprises a cell density of between or between about 5 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, or 15 x 10 ⁶ cells/mL and 25 x 10 ⁶ cells/mL, each inclusive. In some of any embodiments, the input composition comprises a cell density of between or between about 5 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, inclusive. In some of any embodiments, the input composition comprises a cell density of between or between about 10 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, inclusive. In some of any embodiments, the input composition comprises a cell density of between or between about 15 x 10 ⁶ cells/mL and 25 x 10 ⁶ cells/mL, inclusive.

[0128] In some of any embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase comprised in the cavity is between or between about 1 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, or 1 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, each inclusive. In some of any embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase comprised in the cavity is between or between about 1 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, inclusive. In some of any embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase comprised in the cavity is between or between about 1 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, inclusive. In some of any embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase comprised in the cavity is between or between about 1 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, inclusive.

[0129] In some of any embodiments, the method further comprises incubating the input composition in the well prior to the collecting.

[0130] In some of any embodiments, the method further comprises incubating the composition in the one or more wells after the adding and prior to the centrifuging.

[0131] In some of any embodiments, the incubating is performed for between or between about 2 minutes and 120 minutes, 2 minutes and 90 minutes, 2 minutes and 60 minutes, or 2 minutes and 30 minutes, each inclusive. In some of any embodiments, the incubating is performed for between or between about 2 minutes and 120 minutes, inclusive. In some of any embodiments, the incubating is performed for between or between about 2 minutes and 90 minutes, inclusive. In some of any embodiments, the incubating is performed for between or between about 2 minutes and 60 minutes, inclusive. In some of any embodiments, the incubating is performed for between or between about 2 minutes and 30 minutes, inclusive.

[0132] In some of any embodiments, all or a portion of the incubating is performed under mixing conditions. In some of any embodiments, all of the incubating is performed under mixing conditions. In some of any embodiments, the mixing conditions comprise shaking the chromatography array at a speed of between or between about 50 revolutions per minute (rpm) and 1000 rpm, 200 rpm and 800 rpm, or 400 rpm and 600 rpm, each inclusive. In some of any embodiments, the mixing conditions comprise shaking the chromatography array at a speed of between or between about 50 rpm and 1000 rpm, inclusive. In some of any embodiments, the mixing conditions comprise shaking the chromatography array at a speed of between or between about 200 rpm and 800 rpm, inclusive. In some of any embodiments, the mixing conditions comprise shaking the chromatography array at a speed of between or between about 400 rpm and 600 rpm, inclusive. [0133] In some of any embodiments, the collecting comprises flow-through of the target cell through the barrier. In some of any embodiments, the collecting comprises centrifuging the chromatography array or applying a vacuum to the chromatography array. In some of any embodiments, the collecting comprises centrifuging the chromatography array. In some of any embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, 100 x g and 1000 x g, 300 x g and 500 x g, or 350 x g and 450 x g, each inclusive. In some of any embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 100 x g and 1000 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 300 x g and 500 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 350 x g and 450 x g, inclusive.

[0134] In some of any embodiments, the target cell expresses the selection marker, and the collecting comprises: binding of the selection agent to the selection marker expressed by the target cell; centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier; and centrifuging the chromatography array for flow-through of the target cell through the barrier. In some of any embodiments, the collecting comprises adding biotin or a biotin analog to the stationary phase prior to the flowthrough of the target cell through the barrier. In some of any embodiments, the target cell expresses the selection marker, and the collecting comprises: binding of the selection agent to the selection marker expressed by the target cell; centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0135] In some of any embodiments, the collecting comprises adding biotin to the stationary phase prior to the flow-through of the target cell through the barrier. In some of any embodiments, the collecting comprises adding D-biotin to the stationary phase prior to the flowthrough of the target cell through the barrier. In some of any embodiments, the collecting comprises adding a biotin analog to the stationary phase prior to the flow-through of the target cell through the barrier. In some of any embodiments, the biotin analog is iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl- HABA.

[0136] In some of any embodiments, the target cell does not express the selection marker, and the collecting comprises: binding of the selection agent to non-target cells expressing the selection marker; and centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0137] In some of any embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, 100 x g and 1000 x g, 300 x g and 500 x g, or 350 x g and 450 x g, each inclusive. In some of any embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 100 x g and 1000 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 300 x g and 500 x g, inclusive. In some of any embodiments, the centrifuging is performed at between or between about 350 x g and 450 x g, inclusive.

[0138] In some of any embodiments, the centrifuging is performed for between or between about 30 seconds and 10 minutes, 2 minutes and 8 minutes, or 4 minutes and 6 minutes, each inclusive. In some of any embodiments, the centrifuging is performed for between or between about 30 seconds and 10 minutes, inclusive. In some of any embodiments, the centrifuging is performed for between or between about 2 minutes and 8 minutes, inclusive. In some of any embodiments, the centrifuging is performed for between or between about 4 minutes and 6 minutes, inclusive.

[0139] In some of any embodiments, the method further comprises sterilizing the multi-well plate prior to the adding.

Brief Description of the Drawings

[0140] FIG. 1 shows cell yield and the percentage of CD3- cells in the negative fraction of cells subjected to chromatography on a provided chromatography array that included an anti- CD3 selection agent. Results across different input cell densities and centrifugation speeds are shown.

[0141] FIG. 2 shows the percentage of CD3- cells in the negative fraction of cells subjected to chromatography on a provided chromatography array that included an anti-CD3 selection agent. Results across different cell-to-resin ratios are shown. [0142] FIG. 3A shows the percentage of Cas3-CD57+ cells in the negative fraction of cells subjected to chromatography on a provided chromatography array that included an anti-CD57 selection agent. For comparison, the percentage of Cas3-CD57+ cells in the negative fraction of cells processed using anti-CD57 microbeads is also shown. FIG. 3B shows cumulative fold expansion over time for the negative fraction cells.

Detailed Description

[0143] Provided herein are chromatography arrays, for instance those that can be used for the isolation of cells. In some embodiments, the cells are isolated based on their expression or lack of expression of a particular selection marker, e.g., a particular cell surface marker. In some embodiments, a target cell that is isolated using the provided chromatography arrays expresses the selection marker, e.g., is part of a positive fraction of cells (positive selection). In some embodiments, a target cell that is isolated using the provided chromatography arrays does not express the selection marker, e.g., is part of a negative fraction of cells (negative selection). Also provided herein are related methods and kits for preparing a chromatography array, including for preparing any of the provided chromatography arrays. Also provided herein are methods of using a chromatography array, e.g., any of the provided chromatography arrays, including methods for isolating a target cell. In some embodiments, the provided chromatography arrays and methods are suitable for automation or semi- automation.

[0144] In some embodiments, the provided chromatography arrays and methods are suitable for performing high-throughput methods for isolating cells. In some embodiments, the provided chromatography arrays and methods are suitable for performing scale-down methods for isolating cells, e.g., methods in which the number of cells processed using the chromatography array, e.g., per well of the chromatography array, is less than the number of cells that would be processed in a full-scale method, for instance less than the number of cells produced by a full- scale cell engineering or manufacturing method and subsequently processed using a full-scale chromatography method. In some embodiments, the provided chromatography arrays and methods lead to isolated cells with improved health, viability, and/or functional activity, relative to other isolation methods, for instance alternative scale-down isolation methods.

[0145] In some embodiments, the provided methods for isolating cells involve the collection of cells from the chromatography array by centrifugation. In some embodiments, the centrifugation of the chromatography array at higher centrifugation speeds results in improved cell yield and low well-to-well variability in the collection of cells.

[0146] In some embodiments, the provided chromatography arrays contain a stationary phase. In some embodiments, the stationary phase includes a non-magnetic or non-magnetizable material. In some embodiments, the stationary phase includes a chromatography matrix containing a chromatography resin. In some aspects, the use of a non-magnetic or non- magnetizable material, e.g., a chromatography resin, in the provided chromatography arrays improves or maintains the health, viability, and/or functional activity of cells isolated using the provided chromatography arrays, for instance according to any of the provided methods for isolating cells. In some aspects, the health, viability, and/or functional activity of the isolated cells is improved relative to cells isolated using other isolation methods, for instance those involving the use of magnetic beads.

[0147] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. 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 that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0148] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. CHROMATOGRAPHY ARRAYS

[0149] Provided herein are chromatography arrays that can be used for the isolation of cells. In some embodiments, the cells are isolated based on their expression or lack of expression of a particular selection marker, e.g., a particular cell surface marker. In some embodiments, the chromatography array can be used for the selection, e.g., positive or negative selection, of cells based on their expression or lack of expression of the selection marker. In some embodiments, the chromatography array can be used for the enrichment of cells expressing the selection marker (positive selection). In some embodiments, the chromatography array can be used for the depletion of cells expressing the selection marker (negative selection). In some embodiments, the chromatography array is suitable for use in high-throughput methods for isolating cells, for instance in high-throughput methods for the selection, enrichment, or depletion of cells.

[0150] In some embodiments, the chromatography array is sterile.

A. Spatial Arrays

[0151] In some embodiments, the provided chromatography array includes a spatial array of wells. In some embodiments, the spatial array is an array where wells are separated or occupy a distinct space in the array. In some embodiments, the spatial array is an addressable array. In some embodiments, each well of the spatial array is an address in the spatial array. In some embodiments, an address refers to a unique identifier whereby an addressed entity can be identified.

[0152] In some embodiments, each well of the spatial array includes a cavity. In some embodiments, the cavity includes a top opening. In some embodiments, the cavity includes a bottom opening. In some embodiments, the well includes a side wall that partially encloses the cavity. In some embodiments, each well has a volume of between or between about 0.2 mL and 20 mL, 0.2 mL and 18 mL, 0.2 mL and 16 mL, 0.2 mL and 14 mL, 0.2 mL and 12 mL, 0.2 mL and 10 mL, 0.2 mL and 8 mL, 0.2 mL and 6 mL, 0.2 mL and 4 mL, 0.2 mL and 2 mL, 0.2 mL and 1.5 mL, 0.2 mL and 1 mL, 0.2 mL and 0.5 mL, 0.5 mL and 20 mL, 0.5 mL and 18 mL, 0.5 mL and 16 mL, 0.5 mL and 14 mL, 0.5 mL and 12 mL, 0.5 mL and 10 mL, 0.5 mL and 8 mL, 0.5 mL and 6 mL, 0.5 mL and 4 mL, 0.5 mL and 2 mL, 0.5 mL and 1.5 mL, 0.5 mL and 1 mL, 1 mL and 20 mL, 1 mL and 18 mL, 1 mL and 16 mL, 1 mL and 14 mL, 1 mL and 12 mL, 1 mL and 10 mL, 1 mL and 8 mL, 1 mL and 6 mL, 1 mL and 4 mL, 1 mL and 2 mL, 1 mL and 1.5 mL, 1.5 mL and 20 mL, 1.5 mL and 18 mL, 1.5 mL and 16 mL, 1.5 mL and 14 mL, 1.5 mL and 12 mL, 1.5 mL and 10 mL, 1.5 mL and 8 mL, 1.5 mL and 6 mL, 1.5 mL and 4 mL, 1.5 mL and 2 mL, 2 mL and 20 mL, 2 mL and 18 mL, 2 mL and 16 mL, 2 mL and 14 mL, 2 mL and 12 mL, 2 mL and 10 mL, 2 mL and 8 mL, 2 mL and 6 mL, 2 mL and 4 mL, 4 mL and 20 mL, 4 mL and 18 mL, 4 mL and 16 mL, 4 mL and 14 mL, 4 mL and 12 mL, 4 mL and 10 mL, 4 mL and 8 mL, 4 mL and 6 mL, 6 mL and 20 mL, 6 mL and 18 mL, 6 mL and 16 mL, 6 mL and 14 mL, 6 mL and 12 mL, 6 mL and 10 mL, 6 mL and 8 mL, 8 mL and 20 mL, 8 mL and 18 mL, 8 mL and 16 mL, 8 mL and 14 mL, 8 mL and 12 mL, 8 mL and 10 mL, 10 mL and 20 mL, 10 mL and 18 mL, 10 mL and 16 mL, 10 mL and 14 mL, 10 mL and 12 mL, 12 mL and 20 mL, 12 mL and 18 mL, 12 mL and 16 mL, 12 mL and 14 mL, 14 mL and 20 mL, 14 mL and 18 mL, 14 mL and 16 mL, 16 mL and 20 mL, 16 mL and 18 mL, or 18 mL and 20 mL, each inclusive. The well volume can be the same or different across the wells of the spatial array. In some embodiments, each well has a volume of between or between about 0.2 mL and 20 mL, inclusive. In some embodiments, each well has a volume of between or between about 0.2 mL and 10 mL, inclusive. In some embodiments, each well has a volume of between or between about 0.5 mL and 1.5 mL, inclusive.

[0153] In some embodiments, the volume of the well is dependent on the surface area of the well of the array. For instance, a 24-well plate can have a height of 18.70 mm, a length of 127.80 mm, and a width of 85.60 mm. A recommended working volume for a 24-well plate is 0.5-2.39 mL. It is within the level of a skilled artisan to choose an appropriately sized well array for the isolation.

[0154] In some embodiments, the spatial array includes a number of wells suitable for high- throughput methods for isolating cells. In some embodiments, the spatial array includes, includes about, or includes at least 4, 6, 8, 10, 12, 24, 48, 96, 144, 192, 240, 288, 336, 384, 432, 480, 576, 672, 768, 864, 960, 1056, 1152, 1248, 1344, 1440, or 1536 wells. In some embodiments, the spatial array includes at least 12 wells. In some embodiments, the spatial array includes 24 wells. In some embodiments, the spatial array includes 96 wells. In some embodiments, the spatial array is a multi-well plate. In some embodiments, the spatial array is a 4-well, 6-well, 8-well, 10-well, 12-well, 24-well, 48-well, 96-well, 144-well, 192-well, 240- well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plate. In some embodiments, the spatial array includes a plurality of sub-arrays. For example, multiple 96-well plates can constitute a plurality of sub-arrays and a single spatial array.

[0155] In some embodiments, the cavity of each of one or more wells of the spatial array includes, e.g., contains, a stationary phase. Exemplary stationary phases are described in Section LB. In some embodiments, the stationary phase is any as described in Section LB. In some embodiments, the one or more wells with cavities that include the stationary phase include only a subset of wells of the spatial array. In some embodiments, the one or more wells include, include about, or include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 96, 144, 192, 240, 288, 336, 384, 432, 480, 576, 672, 768, 864, 960, 1056, 1152, 1248, 1344, 1440, or 1536 wells of the spatial array. In some embodiments, the one or more wells include all wells of the spatial array. [0156] In some embodiments, each of the one or more wells that include the stationary phase further includes a barrier, which generally is permeable and permits flow-through or passage of materials through the bottom opening of the cavity. In some embodiments, each well of the spatial array includes a barrier. In some embodiments, the barrier is positioned proximal to the bottom opening of the well. In some embodiments, the barrier is positioned to span the bottom opening. In some embodiments, the barrier is positioned at the bottom opening. In some embodiments, the barrier is positioned flush with the bottom opening. In some embodiments, the barrier is secured to the bottom opening. In some embodiments, the barrier is positioned within the cavity of the well. In some embodiments, the barrier is secured within the cavity. In some embodiments, the barrier is secured to the side wall of the cavity.

[0157] In some embodiments, the barrier is permeable to cells. In some embodiments, the barrier is not permeable to the stationary phase. In some embodiments, the barrier is permeable to prokaryotic and eukaryotic cells. In some embodiments, the barrier is permeable to eukaryotic cells. In some embodiments, the barrier is permeable to mammalian cells. In some embodiments, the barrier is permeable to immune cells. In some embodiments, the barrier is permeable to lymphocytes. In some embodiments, the barrier is permeable to T cells, B cells, and/or natural killer (NK) cells. In some embodiments, the barrier is permeable to T cells.

[0158] In some embodiments, the barrier is permeable to peripheral blood mononuclear cells (PBMCs). In some embodiments, the barrier is permeable to B cells. In some embodiments, the barrier is permeable to NK cells. In some embodiments, the barrier is permeable to T cells.

[0159] In some embodiments, the barrier includes pores of sufficient size to allow the passage, e.g., flow-through, of cells. In some embodiments, the barrier includes pores that are at least or at least about 0.1, 0.25, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 pm in diameter. In some embodiments, the barrier includes pores that are between or between about 0.1 pm and 160 pm, 0.1 pm and 140 pm, 0.1 pm and 120 pm, 0.1 pm and 100 pm, 0.1 pm and 80 pm, 0.1 pm and 60 pm, 0.1 pm and 40 pm, 0.1 pm and 20 pm, 0.1 pm and 10 pm, 0.1 pm and 5 pm, 0.1 pm and 1 pm, 0.1 pm and 0.5 pm, 0.5 pm and 160 pm, 0.5 pm and 140 pm, 0.5 pm and 120 pm, 0.5 pm and 100 pm, 0.5 pm and 80 pm, 0.5 pm and 60 pm, 0.5 pm and 40 pm, 0.5 pm and 20 pm, 0.5 pm and 10 pm, 0.5 pm and 5 pm, 0.5 pm and 1 pm, 1 pm and 160 pm, 1 pm and 140 pm, 1 pm and 120 pm, 1 pm and 100 pm, 1 pm and 80 pm, 1 pm and 60 pm, 1 pm and 40 pm, 1 pm and 20 pm, 1 pm and 10 pm, 1 pm and 5 pm, 5 pm and 160 pm, 5 pm and 140 |im, 5 |im and 120 |im, 5 |im and 100 |im, 5 |im and 80 |im, 5 jam and 60 |im, 5 |im and 40 |im, 5 |im and 20 |im, 5 |im and 10 |im, 10 |im and 160 |im, 10 |im and 140 |im, 10 |im and 120 |im, 10 |im and 100 |im, 10 |im and 80 |im, 10 |im and 60 |im, 10 jam and 40 |im, 10 |im and 20 |im, 20 |im and 160 |im, 20 |im and 140 |im, 20 |im and 120 |im, 20 |im and 100 |im, 20 |im and 80 |im, 20 |im and 60 |im, 20 |im and 40 |im, 40 |im and 160 |im, 40 |im and 140 |im, 40 |im and 120 |im, 40 |im and 100 |im, 40 |im and 80 |im, 40 |im and 60 |im, 60 |im and 140 |im, 60 |im and 120 |im, 60 |im and 100 |im, 60 |im and 80 |im, 80 |im and 140 |im, 80 |im and 120 |im, 80 |im and 100 |im, 100 |im and 140 |im, 100 |im and 120 |im, or 120 |im and 140 |im, each inclusive. In some embodiments, the barrier includes pores that are between or between about 10 pm and 150 pm in diameter. In some embodiments, the barrier includes pores that are between or between about 15 pm and 100 pm in diameter. In some embodiments, the barrier includes pores that are between or between about 20 pm and 50 pm in diameter.

[0160] In some embodiments, the barrier includes pores that are between or between about 30 pm and 40 pm in diameter.

[0161] In some embodiments, the pores are of a size to not allow the passage, e.g., flow- through, of the stationary phase.

[0162] Suitable materials for the barrier are known in the art. In some embodiments, the material of the barrier does not affect the health or viability of cells during passage, e.g., flowthrough, of the cells through the barrier. In some embodiments, the barrier includes nylon mesh. In some embodiments, the barrier includes polypropylene and/or polyethylene media. In some embodiments, the barrier includes polypropylene/polyethylene media. In some embodiments, the barrier includes non-woven polypropylene/polyethylene media.

[0163] In some embodiments, the barrier includes polypropylene media. In some embodiments, the barrier includes polyethylene media. In some embodiments, the barrier includes polypropylene and polyethylene media.

[0164] In some embodiments, the barrier is a filter. In some embodiments, the spatial array is a multi- well filter plate. Multi- well filter plates suitable for use in the provided chromatography array are commercially available and include AcroPrep™ (Pall Corporation), Captiva (Agilent Technologies), Nunc™ (ThermoFisher Scientific), MultiScreen-MESH (Millipore Sigma), and Whatman® UNIFILTER® (Cytiva) 96-Well Filter Plates. [0165] In some embodiments, the spatial array, e.g., multi-well plate, has been sterilized. In some of any embodiments, the spatial array, e.g., multi-well plate, is made of material that can be sterilized. In some embodiments, the sterilizing is by methods involving wet heat (e.g., autoclaving), dry heat (e.g., flaming or baking), the use of solvents (e.g., disinfectants, such as alcohol, e.g., ethanol or isopropanol), radiation (e.g., UV, x-ray, or gamma irradiation), and/or gas sterilization (e.g., with ethylene oxide). In some embodiments, the sterilizing involves autoclaving the spatial array, e.g., multi-well plate. In some embodiments, the spatial array, e.g., multi-well plate, has been autoclaved. In some embodiments, the spatial array, e.g., multi-well plate, has been gamma irradiated.

B. Stationary Phases

[0166] In some embodiments, the stationary phase in the cavity of one or more wells of the spatial array includes, e.g., is composed of, a chromatography matrix. Exemplary chromatography matrices are described in Section I-B-l. In some embodiments, the chromatography matrix is any as described in Section I-B-l. In some embodiments, the chromatography matrix has immobilized thereon a selection agent that specifically binds to a selection marker expressed on the surface of a cell. Exemplary selection agents are described in Sections I-B-2 and I-B-3. In some embodiments, the selection agent is any as described in Sections I-B-2 and I-B-3.

[0167] In some embodiments, the selection agent is the same across the one or more wells. In some embodiments, the selection agent is different across the one or more wells. In some embodiments, the different selection agents specifically bind to different selection markers, for instance some wells including a first selection agent binding to a first selection marker, and other wells including a second selection agent binding to a second selection marker that is different from the first selection marker. In some embodiments, the different selection agents specifically bind to the same selection marker. As an example, the different selection agents may bind at different epitopes of the selection marker, or may include different formats, such as a monovalent antibody fragment in one set of wells and a divalent antibody fragment in a second set of wells.

[0168] In some embodiments, the amount of stationary phase included in each cavity is to achieve a desired binding capacity for cells expressing the selection marker. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 0.1 x 10 ⁶ cells and 20 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 18 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 16 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 14 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 12 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 10 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 8 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 6 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 4 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 2 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 1 x 10 ⁶ cells, 1 x 10 ⁶ cells and 20 x 10 ⁶ cells, 1 x 10 ⁶ cells and 18 x 10 ⁶ cells, 1 x 10 ⁶ cells and 16 x 10 ⁶ cells, 1 x 10 ⁶ cells and 14 x 10 ⁶ cells, 1 x 10 ⁶ cells and 12 x 10 ⁶ cells, 1 x 10 ⁶ cells and 10 x 10 ⁶ cells, 1 x 10 ⁶ cells and 8 x 10 ⁶ cells, 1 x 10 ⁶ cells and 6 x 10 ⁶ cells, 1 x 10 ⁶ cells and 4 x

10 ⁶ cells, 1 x 10 ⁶ cells and 2 x 10 ⁶ cells, 2 x 10 ⁶ cells and 20 x 10 ⁶ cells, 2 x 10 ⁶ cells and 18 x

10 ⁶ cells, 2 x 10 ⁶ cells and 16 x 10 ⁶ cells, 2 x 10 ⁶ cells and 14 x 10 ⁶ cells, 2 x 10 ⁶ cells and 12 x

10 ⁶ cells, 2 x 10 ⁶ cells and 10 x 10 ⁶ cells, 2 x 10 ⁶ cells and 8 x 10 ⁶ cells, 2 x 10 ⁶ cells and 6 x 10 ⁶ cells, 2 x 10 ⁶ cells and 4 x 10 ⁶ cells, 4 x 10 ⁶ cells and 20 x 10 ⁶ cells, 4 x 10 ⁶ cells and 18 x 10 ⁶ cells, 4 x 10 ⁶ cells and 16 x 10 ⁶ cells, 4 x 10 ⁶ cells and 14 x 10 ⁶ cells, 4 x 10 ⁶ cells and 12 x 10 ⁶ cells, 4 x 10 ⁶ cells and 10 x 10 ⁶ cells, 4 x 10 ⁶ cells and 8 x 10 ⁶ cells, 4 x 10 ⁶ cells and 6 x 10 ⁶ cells, 6 x 10 ⁶ cells and 20 x 10 ⁶ cells, 6 x 10 ⁶ cells and 18 x 10 ⁶ cells, 6 x 10 ⁶ cells and 16 x 10 ⁶ cells, 6 x 10 ⁶ cells and 14 x 10 ⁶ cells, 6 x 10 ⁶ cells and 12 x 10 ⁶ cells, 6 x 10 ⁶ cells and 10 x 10 ⁶ cells, 6 x 10 ⁶ cells and 8 x 10 ⁶ cells, 8 x 10 ⁶ cells and 20 x 10 ⁶ cells, 8 x 10 ⁶ cells and 18 x 10 ⁶ cells, 8 x 10 ⁶ cells and 16 x 10 ⁶ cells, 8 x 10 ⁶ cells and 14 x 10 ⁶ cells, 8 x 10 ⁶ cells and 12 x 10 ⁶ cells, 8 x 10 ⁶ cells and 10 x 10 ⁶ cells, 10 x 10 ⁶ cells and 20 x 10 ⁶ cells, 10 x 10 ⁶ cells and 18 x 10 ⁶ cells, 10 x 10 ⁶ cells and 16 x 10 ⁶ cells, 10 x 10 ⁶ cells and 14 x 10 ⁶ cells, 10 x 10 ⁶ cells and 12 x 10 ⁶ cells, 12 x 10 ⁶ cells and 20 x 10 ⁶ cells, 12 x 10 ⁶ cells and 18 x 10 ⁶ cells, 12 x 10 ⁶ cells and 16 x 10 ⁶ cells, 12 x 10 ⁶ cells and 14 x 10 ⁶ cells, 14 x 10 ⁶ cells and 20 x 10 ⁶ cells, 14 x 10 ⁶ cells and 18 x 10 ⁶ cells, 14 x 10 ⁶ cells and 16 x 10 ⁶ cells, 16 x 10 ⁶ cells and 20 x 10 ⁶ cells, 16 x 10 ⁶ cells and 18 x 10 ⁶ cells, or 18 x 10 ⁶ cells and 20 x 10 ⁶ cells expressing the selection marker, each inclusive. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 20 x 10 ⁶ cells expressing the selection marker.

[0169] In some embodiments, the amount of stationary phase included in each cavity is to achieve a desired binding capacity for cells expressing the selection marker. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 0.1 x 10 ⁶ cells and 10 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 9 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 8 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 7 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 6 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 5 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 4 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 3 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 2 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 1 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 0.5 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 10 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 9 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 8 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 7 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 6 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 5 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 4 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 3 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 2 x 10 ⁶ cells, or 0.5 x 10 ⁶ cells and 1 x 10 ⁶ cells expressing the selection marker, each inclusive. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells, 1 x 10 ⁶ and 9 x 10 ⁶ cells, 1 x 10 ⁶ and 8 x 10 ⁶ cells, 1 x 10 ⁶ and 7 x 10 ⁶ cells, 1 x 10 ⁶ and 6 x 10 ⁶ cells, 1 x 10 ⁶ and 5 x 10 ⁶ cells, 1 x 10 ⁶ and 4 x 10 ⁶ cells, 1 x 10 ⁶ and 3 x 10 ⁶ cells, 1 x 10 ⁶ and 2 x 10 ⁶ cells, 2 x 10 ⁶ and 10 x 10 ⁶ cells, 2 x 10 ⁶ and 9 x 10 ⁶ cells, 2 x 10 ⁶ and 8 x 10 ⁶ cells, 2 x 10 ⁶ and 7 x 10 ⁶ cells, 2 x 10 ⁶ and 6 x 10 ⁶ cells, 2 x 10 ⁶ and 5 x 10 ⁶ cells, 2 x 10 ⁶ and 4 x 10 ⁶ cells, 2 x 10 ⁶ and 3 x 10 ⁶ cells, 3 x 10 ⁶ and 10 x 10 ⁶ cells, 3 x 10 ⁶ and 9 x 10 ⁶ cells, 3 x 10 ⁶ and 8 x 10 ⁶ cells, 3 x 10 ⁶ and 7 x 10 ⁶ cells, 3 x 10 ⁶ and 6 x 10 ⁶ cells, 3 x 10 ⁶ and 5 x 10 ⁶ cells, 3 x 10 ⁶ and 4 x 10 ⁶ cells, 4 x 10 ⁶ and 10 x 10 ⁶ cells, 4 x 10 ⁶ and 9 x 10 ⁶ cells, 4 x 10 ⁶ and 8 x 10 ⁶ cells, 4 x 10 ⁶ and 7 x 10 ⁶ cells, 4 x 10 ⁶ and 6 x 10 ⁶ cells, 4 x 10 ⁶ and 5 x 10 ⁶ cells, 5 x 10 ⁶ and 10 x 10 ⁶ cells, 5 x 10 ⁶ and 9 x 10 ⁶ cells, 5 x 10 ⁶ and 8 x 10 ⁶ cells, 5 x 10 ⁶ and 7 x 10 ⁶ cells, 5 x 10 ⁶ and 6 x 10 ⁶ cells, 6 x 10 ⁶ and 10 x 10 ⁶ cells, 6 x 10 ⁶ and 9 x 10 ⁶ cells, 6 x 10 ⁶ and 8 x 10 ⁶ cells, 6 x 10 ⁶ and 7 x 10 ⁶ cells, 7 x 10 ⁶ and 10 x 10 ⁶ cells, 7 x 10 ⁶ and 9 x 10 ⁶ cells, 7 x 10 ⁶ and 8 x 10 ⁶ cells, 8 x 10 ⁶ and 10 x 10 ⁶ cells, 8 x 10 ⁶ and 9 x 10 ⁶ cells, or 9 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker, each inclusive. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker.

[0170] In some embodiments, the amount of stationary phase included in each cavity between or between about 10 mg and 10,000 mg, 10 mg and 9,000 mg, 10 mg and 8,000 mg, 10 mg and 7,000 mg, 10 mg and 6,000 mg, 10 mg and 5,000 mg, 10 mg and 4,000 mg, 10 mg and 3,000 mg, 10 mg and 2,000 mg, 10 mg and 1,000 mg, 10 mg and 500 mg, 500 mg and 10,000 mg, 500 mg and 9,000 mg, 500 mg and 8,000 mg, 500 mg and 7,000 mg, 500 mg and 6,000 mg, 500 mg and 5,000 mg, 500 mg and 4,000 mg, 500 mg and 3,000 mg, 500 mg and 2,000 mg, 500 mg and 1,000 mg, 1,000 mg and 10,000 mg, 1,000 mg and 9,000 mg, 1,000 mg and 8,000 mg, 1,000 mg and 7,000 mg, 1,000 mg and 6,000 mg, 1,000 mg and 5,000 mg, 1,000 mg and 4,000 mg, 1,000 mg and 3,000 mg, 1,000 mg and 2,000 mg, 2,000 mg and 10,000 mg, 2,000 mg and 9,000 mg, 2,000 mg and 8,000 mg, 2,000 mg and 7,000 mg, 2,000 mg and 6,000 mg, 2,000 mg and 5,000 mg, 2,000 mg and 4,000 mg, 2,000 mg and 3,000 mg, 3,000 mg and 10,000 mg, 3,000 mg and 9,000 mg, 3,000 mg and 8,000 mg, 3,000 mg and 7,000 mg, 3,000 mg and 6,000 mg, 3,000 mg and 5,000 mg, 3,000 mg and 4,000 mg, 4,000 mg and 10,000 mg, 4,000 mg and 9,000 mg, 4,000 mg and 8,000 mg, 4,000 mg and 7,000 mg, 4,000 mg and 6,000 mg, 4,000 mg and 5,000 mg, 5,000 mg and 10,000 mg, 5,000 mg and 9,000 mg, 5,000 mg and 8,000 mg, 5,000 mg and 7,000 mg, 5,000 mg and 6,000 mg, 6,000 mg and 10,000 mg, 6,000 mg and 9,000 mg, 6,000 mg and 8,000 mg, 6,000 mg and 7,000 mg, 7,000 mg and 10,000 mg, 7,000 mg and 9,000 mg, 7,000 mg and 8,000 mg, 8,000 mg and 10,000 mg, 8,000 mg and 9,000 mg, or 9,000 mg and 10,000 mg, each inclusive. In some embodiments, the amount of stationary phase included in each cavity between or between about 10 mg and 10,000 mg, inclusive. In some embodiments, the amount of stationary phase included in each cavity between or between about 10 mg and 4,000 mg, inclusive.

[0171] In some embodiments, the amount of stationary phase included in each cavity is between or between about 10 mg and 3000 mg, 10 mg and 2500 mg, 10 mg and 2000 mg, 10 mg and 1500 mg, 10 mg and 1000 mg, 10 mg and 800 mg, 10 mg and 600 mg, 10 mg and 400 mg, 10 mg and 200 mg, 10 mg and 100 mg, 100 mg and 3000 mg, 100 mg and 2500 mg, 100 mg and 2000 mg, 100 mg and 1500 mg, 100 mg and 1000 mg, 100 mg and 800 mg, 100 mg and 600 mg, 100 mg and 400 mg, 100 mg and 200 mg, 200 mg and 3000 mg, 200 mg and 2500 mg, 200 mg and 2000 mg, 200 mg and 1500 mg, 200 mg and 1000 mg, 200 mg and 800 mg, 200 mg and 600 mg, 200 mg and 400 mg, 400 mg and 3000 mg, 400 mg and 2500 mg, 400 mg and 2000 mg, 400 mg and 1500 mg, 400 mg and 1000 mg, 400 mg and 800 mg, 400 mg and 600 mg, 600 mg and 3000 mg, 600 mg and 2500 mg, 600 mg and 2000 mg, 600 mg and 1500 mg, 600 mg and 1000 mg, 600 mg and 800 mg, 800 mg and 3000 mg, 800 mg and 2500 mg, 800 mg and 2000 mg, 800 mg and 1500 mg, 800 mg and 1000 mg, 1000 mg and 3000 mg, 1000 mg and 2500 mg, 1000 mg and 2000 mg, 1000 mg and 1500 mg, 1500 mg and 3000 mg, 1500 mg and 2500 mg, 1500 mg and 2000 mg, 2000 mg and 3000 mg, 2000 mg and 2500 mg, 2500 mg and 3000 mg, each inclusive. In some embodiments, the amount of stationary phase included in each cavity is between or between about 10 mg and 3000 mg, inclusive. In some embodiments, the amount of stationary phase included in each cavity is between or between about 10 mg and 1000 mg, inclusive. In some embodiments, the amount of stationary phase included in each cavity is between or between about 100 mg and 500 mg, inclusive. In some embodiments, the amount of stationary phase included in each cavity is between or between about 200 mg and 400 mg, inclusive.

J. Chromatography Matrices

[0172] Materials known to be suitable for the chromatographic isolation of cells can be employed as part of the chromatography matrix included in the provided chromatography array. In some embodiments, the chromatography matrix is essentially innocuous, e.g., is not detrimental to the health or viability of cells added to the chromatography matrix. In some embodiments, the chromatography matrix includes a non-magnetic material or non- magnetizable material. In some embodiments, the chromatography matrix includes a monolithic matrix. In some embodiments, the chromatography matrix includes a membrane matrix. In some embodiments, the chromatography matrix includes a particulate matrix. In some embodiments, the chromatography matrix includes a beaded matrix.

[0173] In some embodiments, the chromatography matrix includes derivatized silica or a crosslinked gel. In some embodiments, the crosslinked gel is based on a natural polymer, for instance a polysaccharide. In some embodiments, the polysaccharide is crosslinked. Examples of a polysaccharide matrix include an agarose gel (for example, Superflow™ agarose or a Sepharose® material such as Superflow™ Sepharose® that is commercially available in different bead and pore sizes) or a gel of crosslinked dextrans. Further examples include a particulate cross-linked agarose matrix to which dextran is covalently bonded, for instance that is commercially available (in various bead sizes and with various pore sizes) as Sephadex® or Superdex®, both available from GE Healthcare. Further examples include Sephacryl® which is also available in different bead and pore sizes from GE Healthcare.

[0174] In some embodiments, the crosslinked gel is based on a synthetic polymer. In some embodiments, the synthetic polymer is a polymer that has polar monomer units and which is therefore in itself polar. In some embodiments, the synthetic polymer is hydrophilic. Examples of synthetic polymers include polyacrylamides, a styrene-divinylbenzene gel, and a copolymer of an acrylate and a diol or of an acrylamide and a diol. An illustrative example is a polymethacrylate gel, commercially available as a Fractogel®. A further example is a copolymer of ethylene glycol and methacrylate, commercially available as a Toyopearl®. In some embodiments, the chromatography matrix includes natural and synthetic polymer components, such as a composite matrix or a composite or a co-polymer of a polysaccharide and agarose, e.g., a polyacrylamide/agarose composite, or of a polysaccharide and N,N’- methylenebisacrylamide. An illustrative example of a copolymer of a dextran and N,N’- methylenebisacrylamide is the Sephacryl® series of material. A derivatized silica may include silica particles that are coupled to a synthetic or to a natural polymer. Examples of such embodiments include polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N- isopropylacrylamide) grafted silica.

[0175] In some embodiments, the chromatography matrix includes a particulate matrix. In some embodiments, the chromatography matrix includes a polymeric resin, metal oxide, metalloid oxide, or mixed oxide. In some embodiments, particulates of the particulate matrix have a mean particle size of between or between about 5 pm and 600 pm, 5 pm and 400 pm, 5 pm and 200 pm, 5 pm and 150 pm, 5 pm and 125 pm, 5 pm and 100 pm, 5 pm and 75 pm, 5 pm and 50 pm, 5 pm and 25 pm, 25 pm and 600 pm, 25 pm and 400 pm, 25 pm and 200 pm, 25 pm and 150 pm, 25 pm and 125 pm, 25 pm and 100 pm, 25 pm and 75 pm, 25 pm and 50 pm, 50 pm and 600 pm, 50 pm and 400 pm, 50 pm and 200 pm, 50 pm and 150 pm, 50 pm and 125 pm, 50 pm and 100 pm, 50 pm and 75 pm, 75 pm and 600 pm, 75 pm and 400 pm, 75 pm and 200 pm, 75 pm and 150 pm, 75 pm and 125 pm, 75 pm and 100 pm, 100 pm and 600 pm, 100 pm and 400 pm, 100 pm and 200 pm, 100 pm and 150 pm, 100 pm and 125 pm, 125 pm and 600 pm, 125 pm and 400 pm, 125 pm and 200 pm, 125 pm and 150 pm, 150 pm and 600 pm, 150 pm and 400 pm, 150 pm and 200 pm, 200 pm and 600 pm, 200 pm and 400 pm, or 400 pm and 600 pm, each inclusive. In some embodiments, the chromatography resin beads are between or between about 50 pm and 150 pm in diameter, inclusive. In some embodiments, the chromatography resin beads are between or between about 75 pm and 125 pm in diameter, inclusive. In some embodiments, the chromatography resin beads are between or between about 90 pm and 110 pm in diameter, inclusive.

[0176] In some embodiments, the chromatography matrix includes a chromatography resin. In some embodiments, the chromatography matrix includes chromatography resin beads, such as those commercially available as CytoSorb® (CytoSorbents™). In some embodiments, the resin includes a polystyrene resin. In some embodiments, the chromatography resin beads are between or between about 5 pm and 600 |im, 5 |im and 400 |im, 5 |im and 200 |im, 5 |im and 150 |im, 5 jam and 125 |im, 5 |im and 100 |im, 5 |im and 75 |im, 5 |im and 50 |im, 5 |im and 25 |im, 25 |im and 600 |im, 25 |im and 400 |im, 25 |im and 200 |im, 25 |im and 150 |im, 25 |im and 125 |im, 25 |im and 100 |im, 25 |im and 75 |im, 25 |im and 50 |im, 50 |im and 600 |im, 50 |im and 400 |im, 50 |im and 200 |im, 50 |im and 150 |im, 50 |im and 125 |im, 50 |im and 100 |im, 50 |im and 75 |im, 75 |im and 600 |im, 75 |im and 400 |im, 75 |im and 200 |im, 75 |im and 150 |im, 75 |im and 125 |im, 75 |im and 100 |im, 100 |im and 600 |im, 100 |im and 400 |im, 100 |im and 200 |im, 100 |im and 150 |im, 100 |im and 125 |im, 125 |im and 600 |im, 125 |im and 400 |im, 125 jam and 200 |im, 125 |im and 150 |im, 150 |im and 600 |im, 150 |im and 400 |im, 150 |im and 200 |im, 200 |im and 600 |im, 200 |im and 400 |im, or 400 |im and 600 |im in diameter, each inclusive.

2. Selection Agents and Markers

[0177] In some embodiments, the selection marker specifically bound by the selection agent of the stationary phase is a peptide or a protein, such as a membrane receptor protein. The selection marker can be a peripheral membrane protein or an integral membrane protein, in some embodiments having one or more domains that span the membrane. In some embodiments, the selection marker is a lipid, a polysaccharide, or a nucleic acid.

[0178] In some embodiments, the selection marker is a marker expressed by or defining a cell population, for instance a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, B cells, or NK 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 is a marker expressed on the surface of immune cells. In some embodiments, the selection marker is a marker expressed on the surface of lymphocytes. In some embodiments, the selection marker is a marker expressed on the surface of T cells, B cells, or NK cells. In some embodiments, the selection marker is a marker expressed on the surface of T cells. Examples of T cells include cells such as CMV-specific CD8+ T cells, 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. In some embodiments, the selection marker is a T cell coreceptor. In some embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, and/or CD45RO. [0179] In some embodiments, the selection agent includes an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an MHC molecules, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing, that specifically binds to the selection marker. In some embodiments, the selection agent includes an antibody fragment. Exemplary antibody fragments include Fab fragments, Fv fragments, single-chain Fv fragments (scFv), divalent antibody fragments such as F(ab’)2-fragments, diabodies, triabodies (Iliades, P., et al., FEBS Let (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, E.J., et al., Trends Biotechnol. (2003), 21, 11, 484-490).

[0180] In some embodiments, the selection agent binds to the selection marker in a monovalent manner. Examples of monovalent selection agents include a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an aptamer, and an MHC molecule. In some embodiments, the selection agent includes a monovalent antibody fragment. In some embodiments, the monovalent antibody fragment is a Fab fragment, Fv fragment, or single-chain Fv fragment (scFv). In some embodiments, the monovalent antibody fragment is a Fab fragment.

[0181] In some embodiments, the selection agent includes an antibody fragment that is a divalent antibody fragment. In some embodiments, the divalent antibody fragment is an F(ab’)2- fragment or a divalent single-chain Fv fragment.

[0182] In some embodiments, the selection agent includes a proteinaceous molecule with antibody-like binding properties. In some embodiments, the proteinaceous molecule with antibody-like binding properties is an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, or an avimer. Other exemplary proteinaceous molecules include 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 Gia 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, EDE-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” (cf. Ill. Et al., Protein Eng (1997) 10, 949-57, a so called “minibody” (Martin et al., EMBO J (1994) 13, 5303-5309), a diabody (cf. Holliger et al., PNAS USA (1993)90, 6444-6448), a so called “Janusis” (cf. 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, and a leucine-rich repeat protein. In some embodiments, the selection agent is a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein that is also known as “duocalin”.

[0183] In some embodiments, the selection marker is CD4, and the selection agent specifically binds CD4. In some embodiments, the selection agent includes an anti-CD4- antibody, a divalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4-antibody, or a proteinaceous CD4 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD4-antibody, divalent antibody fragment of an anti-CD4 antibody, or monovalent antibody fragment of an anti-CD4-antibody (e.g., anti- CD4 Fab fragment) is derived from antibody 13B8.2 or a functionally active mutant of 13B8.2 that retains specific binding for CD4. Exemplary mutants of antibody 13B8.2 or ml3B8.2 are described in U.S. Patent Nos. 7,482,000, U.S. Patent Appl. No. US2014/0295458, International Patent Application No. WO2013/124474, and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment termed “ml3B8.2” carries the variable domain of the CD4 binding murine antibody 13B8.2 and a constant domain containing constant human CHI 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 embodiments, 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 selection agent includes an anti-CD4 Fab fragment. In some embodiments, the anti-CD4 Fab fragment includes a variable heavy chain having the sequence set forth by SEQ ID NO: 29 and a variable light chain having the sequence set forth by SEQ ID NO: 30. In some embodiments, the anti-CD4 Fab fragment includes the CDRs of the variable heavy chain having the sequence set forth by SEQ ID NO: 29 and the CDRs of the variable light chain having the sequence set forth by SEQ ID NO: 30.

[0184] In some embodiments, the selection marker is CD8, and the selection agent specifically binds CD8. In some embodiments, the selection agent includes an anti-CD8- antibody, a divalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8-antibody, or a proteinaceous CD8 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD8-antibody, divalent antibody fragment of an anti-CD8 antibody, or monovalent antibody fragment of an anti-CD8-antibody (e.g., anti- CD8 Fab fragment) is derived from antibody OKT8 (e.g., ATCC CRL-8014) or a functionally active mutant thereof that retains specific binding for CD8. In some embodiments, the selection agent includes an anti-CD8 Fab fragment. In some embodiments, the anti-CD8 Fab fragment includes a variable heavy chain having the sequence set forth by SEQ ID NO: 36 and a variable light chain having the sequence set forth by SEQ ID NO: 37. In some embodiments, the anti- CD8 Fab fragment includes the CDRs of the variable heavy chain having the sequence set forth by SEQ ID NO: 36 and the CDRs of the variable light chain having the sequence set forth by SEQ ID NO: 37.

[0185] In some embodiments, the selection marker is CD3, and the selection agent specifically binds CD3. In some embodiments, the selection agent includes an anti-CD3- antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3-antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3-antibody, divalent antibody fragment of an anti-CD3 antibody, or monovalent antibody fragment of an anti-CD3-antibody (e.g., anti- CD3 Fab fragment) is derived from antibody OKT3 (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PloS One. 2012; 7(4): e35798) or a functionally active mutant thereof that retains specific binding for CD3. In some embodiments, the selection agent includes an anti- CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment includes 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. In some embodiments, the anti-CD3 Fab fragment includes the CDRs of the variable heavy chain having the sequence set forth by SEQ ID NO: 31 and the CDRs of the variable light chain having the sequence set forth by SEQ ID NO: 32.

[0186] In some embodiments, the selection marker is CD25, and the selection agent specifically binds CD25. In some embodiments, the selection agent includes an anti-CD25 antibody, a divalent antibody fragment of an anti-CD25 antibody, a monovalent antibody fragment of an anti-CD25 antibody, or a proteinaceous CD25 binding molecule with antibodylike binding properties. In some embodiments, the anti-CD25 antibody, divalent antibody fragment of an anti-CD25 antibody, or monovalent antibody fragment of an anti-CD25 antibody (e.g., anti-CD25 Fab fragment) is derived from antibody FRT5 (see, e.g., Stemberger et al. 2012. PloS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding for CD25.

[0187] In some embodiments, the selection marker is CD62L, and the selection agent specifically binds CD62L. In some embodiments, the selection agent includes an anti-CD62L antibody, a divalent antibody fragment of an anti-CD62L antibody, a monovalent antibody fragment of an anti-CD62L antibody, or a proteinaceous CD62L binding molecule with antibody-like binding properties. In some embodiments, the anti-CD62L antibody, divalent antibody fragment of an anti-CD62L antibody, or monovalent antibody fragment of an anti- CD62L antibody (e.g., anti-CD62L Fab fragment) is derived from antibody DREG56 (e.g., ATCC HB300; see, e.g., Stemberger et al. 2012, PloS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding for CD62L.

[0188] In some embodiments, the selection marker is CD45RA, and the selection agent specifically binds CD45RA. In some embodiments, the selection agent includes an anti- CD45RA antibody, a divalent antibody fragment of an anti-CD45RA antibody, a monovalent antibody fragment of an anti-CD45RA antibody, or a proteinaceous CD45RA binding molecule with antibody-like binding properties. In some embodiments, the anti-CD45RA antibody, divalent antibody fragment of an anti-CD45RA antibody, or monovalent antibody fragment of an anti-CD45RA antibody (e.g., anti-CD45RA Fab fragment) is derived from antibody MEM56 (e.g., Millipore 05-1413; see, e.g., Stemberger et al. 2012, PloS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding for CD45RA. 3. Protein Peagents and Binding Partners

[0189] In some embodiments, the selection agent of the stationary phase is bound covalently or non-covalently to the chromatography matrix. In some embodiments, the selection agent is immobilized to the chromatography matrix via a reagent attached to the chromatography matrix. Thus, in some embodiments, the selection agent is bound to the attached reagent, and the selection agent is indirectly immobilized to the chromatography matrix. In some embodiments, the selection agent is reversibly immobilized to the chromatography matrix, for instance such that immobilization of the selection agent on the stationary phase is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some embodiments, the competition reagent has higher affinity for the attached reagent than does the selection agent, thereby outcompeting the selection agent for binding to the attached reagent.

[0190] In some embodiments, the selection agent includes a binding partner that is bound to the attached reagent. In some embodiments, the binding partner is hydrocarbon-based (including polymeric) and includes nitrogen-, phosphorus-, sulphur-, carben-, halogen- or pseudohalogen groups. In some embodiments, the binding partner is 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. In some embodiments, the binding partner is a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube, or carbon nanofoam.

[0191] In some embodiments, the binding partner includes a moiety known to the skilled artisan as an affinity tag. In some embodiments, the attached reagent includes 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 that is included in the selection 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 (e.g., SEQ ID NO: 20), the VSV-G-tag (e.g., SEQ ID NO: 21), the HSV-tag (e.g., SEQ ID NO: 22), the T7 epitope (e.g., SEQ ID NO: 23), maltose binding protein (MBP), the HSV epitope of the sequence of herpes simplex virus glycoprotein D (e.g., SEQ ID NO: 24), the “myc” epitope of the transcription factor c-myc of the sequence (e.g., SEQ ID NO: 25), the V5- tag (e.g., SEQ ID NO: 26), the MAT tag (e.g., SEQ ID NO: 35), or glutathione-S -transferase (GST).

[0192] In some embodiments, the attached reagent is a protein reagent. In some embodiments, the protein reagent includes streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing. In some embodiments, the binding partner of the selection agent is bound to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture. In some embodiments, the binding partner is bound to the biotin-binding pocket of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

[0193] In some embodiments, the protein reagent includes streptavidin or a streptavidin mutein. In some embodiments, the binding partner of the selection agent is bound to the streptavidin or streptavidin mutein. In some embodiments, the binding partner is bound to the biotin-binding pocket of the streptavidin or streptavidin mutein.

[0194] In some embodiments, the protein reagent includes a streptavidin mutein. In some embodiments, the binding partner of the selection agent is bound to the streptavidin mutein. In some embodiments, the binding partner is bound to the biotin-binding pocket of the streptavidin mutein.

[0195] In some embodiments, the binding partner is reversibly bound to the protein reagent. In some embodiments, the binding of the binding partner to the protein reagent effects the reversible immobilization of the selection agent. In some embodiments, the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase. In some embodiments, the competition reagent has higher affinity for the protein reagent than does the selection agent, thereby outcompeting the selection agent for binding to the protein reagent. In some embodiments, the competition reagent includes biotin or a biotin analog. Exemplary biotin analogs include desthiobiotin, iminobiotin, and diaminobiotin.

[0196] Further exemplary biotin analogs include lipoic acid, hydroxyazobenzene-benzoic acid (HABA), and dimethyl-HABA.

[0197] In some embodiments, the streptavidin mutein includes a polypeptide sequence distinguished from the sequence of a wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but that includes a binding site for biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, wild-type streptavidin has the amino acid sequence disclosed by Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882 (SEQ ID NO: 1). In some embodiments, the streptavidin mutein includes only a part of wild-type streptavidin. In some embodiments, the streptavidin mutein includes a minimal streptavidin wherein wild-type streptavidin is shortened at the N- and/or C-terminus. Such minimal streptavidins can include any that begin N-terminally in the region of amino acid positions 10 to 16 of SEQ ID NO: 1 and terminate C-terminally in the region of amino acid positions 133 to 142 of SEQ ID NO: 1. In some embodiments, the minimal streptavidin includes an amino acid sequence from position Alal3 to Serl39 of SEQ ID NO: 1. In some embodiments, the minimal streptavidin further includes an N-terminal methionine at a position corresponding to Alal3 of SEQ ID NO: 1. In some embodiments, the minimal streptavidin includes the sequence of amino acids set forth in SEQ ID NO: 2. In some embodiments, the minimal streptavidin includes the sequence of amino acids set forth in SEQ ID NO: 38. Reference to the position of residues in streptavidin or streptavidin muteins is with reference to numbering of residues in SEQ ID NO: 1.

[0198] In some embodiments, the streptavidin mutein includes one or more amino acid substitutions (replacements) compared to wild-type streptavidin, such as compared to the wildtype streptavidin sequence set forth in SEQ ID NO: 1, or compared to a minimal streptavidin, such as compared to the sequence set forth in SEQ ID NO: 2 or 38. In some embodiments, the streptavidin mutein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences compared to the sequence of amino acids set forth in SEQ ID NO: 1, 2, or 38 or includes an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO: 1, 2, or 38, where the streptavidin mutein binds to biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the amino acid replacements (substitutions) are conservative or non-conservative mutations. Examples of streptavidin muteins are known in the art, see, e.g., U.S. Pat. No. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; 6,368,813; and PCT App. No. WO2014/076277.

[0199] In some embodiments, the streptavidin mutein is as described in U.S. Pat. No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within the region of amino acid positions 44 to 53, based on the amino acid sequence of wild-type streptavidin, such as set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains a mutation at one or more residues 44, 45, 46, and/or 47. In some embodiments, the streptavidin mutein contains a replacement of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, e.g., Vai, Ala, He or Leu, any amino acid at position 45, an aliphatic amino acid, such as a hydrophobic aliphatic amino acid at position 46 and/or a replacement of Vai at position 47 with a basic amino acid, e.g., Arg or Lys, such as generally Arg. In some embodiments, Ala is at position 46 and/or Arg is at position 47 and/or Vai or He is at position 44. In some embodiments, the streptavidin mutein contains residues Val ⁴⁴ -Thr ⁴⁵ - Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 42), such as set forth in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO: 3, 4, or 39. In some embodiments, the streptavidin mutein contains residues Ilc^-Gly^-Ala^’-Arg ⁴⁷ (SEQ ID NO: 41), such as set forth in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO: 5, 6, or 40. In some embodiments, such streptavidin muteins are described, for example, in US patent 6,103,493, and are commercially available under the trademark Strep-Tactin®, for instance as Strep-Tactin® m2.

[0200] In some embodiment, the streptavidin mutein is as described in International Published PCT Appl. Nos. WO 2014/076277. In some embodiments, the streptavidin mutein contains at least two cysteine residues in the region of amino acid positions 44 to 53 with reference to amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the cysteine residues are present at positions 45 and 52 to create a disulfide bridge connecting these amino acids. In such an embodiment, amino acid 44 is typically glycine or alanine and amino acid 46 is typically alanine or glycine and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acids residues 115 to 121 with reference to amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid position 117, 120 and 121 and/or a deletion of amino acids 118 and 119 and substitution of at least amino acid position 121.

[0201] In some embodiments, the streptavidin mutein contains a mutation at a position corresponding to position 117, which mutation can be to a large hydrophobic residue like Trp, Tyr or Phe or a charged residue like Glu, Asp or Arg or a hydrophilic residue like Asn or Gin, or, in some cases, the hydrophobic residues Leu, Met or Ala, or the polar residues Thr, Ser or His. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120, which mutation can be to a small residue like Ser or Ala or Gly, and a mutation at a position corresponding to position 121, which mutation can be to a hydrophobic residue, such as a bulky hydrophobic residue like Trp, Tyr or Phe. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 of wildtype streptavidin set forth in SEQ ID NO: 1 or a biologically active fragment thereof, which mutation can be a hydrophobic residue such as Leu, He, Met, or Vai or, generally, Tyr or Phe, and a mutation at a position corresponding to position 121 compared to positions of wildtype streptavidin set forth in SEQ ID NO: 1 or a biologically active fragment thereof, which mutation can be to a small residue like Gly, Ala, or Ser, or with Gin, or with a hydrophobic residue like Leu, Vai, He, Trp, Tyr, Phe, or Met. In some embodiments, such muteins also can contain residues Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ (SEQ ID NO: 42) or residues Ilc^-Gly^-Ala^’-Arg ⁴⁷ (SEQ ID NO: 41). In some embodiments, the streptavidin mutein contains the residues Val44, Thr45, Ala46, Arg47, Glul l7, Glyl20 and Tyrl21. In some embodiments, the mutein streptavidin contains the sequence of amino acids set forth in SEQ ID NO: 27 or SEQ ID NO: 28, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO: 27 or SEQ ID NO: 28, contains the residues Val44, Thr45, Ala46, Arg47, Glul l7, Gly 120 and Tyrl21 and binds to biotin, a biotin analog, or a streptavidin-binding peptide.

[0202] In some embodiments, the protein reagent includes an avidin mutein, such as neutravidin, a deglycosylated avidin with modified arginines that typically exhibits a more neutral pi and is available as an alternative to native avidin. Deglycosylated, neutral forms of avidin include commercially available forms such as “Extravidin”, available through Sigma Aldrich, or “NeutrAvidin”, available from Thermo Scientific or Invitrogen.

[0203] In some embodiments, the binding partner includes biotin, a biotin analog (e.g., desthiobiotin, iminobiotin, or diaminobiotin), or a streptavidin-binding peptide. In some embodiments, the binding partner includes a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide contains 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 has the general formula set forth in SEQ ID NO: 11, such as set forth in SEQ ID NO: 12. In some embodiments, the streptavidin-binding peptide includes Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag®, set forth in SEQ ID NO: 7). In some embodiments, the streptavidin-binding peptide includes 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 contains at least two streptavidin-binding modules. In some embodiments, the streptavidin-binding peptide contains a sequential arrangement of the at least two streptavidin-binding modules. In some embodiments, the distance between the two modules is at least 0 and not greater than 50 amino acids. In some embodiments, 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. In some embodiments, the other binding module has the same or different streptavidin-binding sequence, such as set forth in SEQ ID NO: 11 (see, e.g., International Published PCT Appl. No. W002/077018; U.S. Patent No. 7,981,632). In some embodiments, the streptavidin-binding peptide contains a sequence having the formula set forth in SEQ ID NO: 13 or 14. In some embodiments, the streptavidin-binding peptide includes the sequence of amino acids set forth in any of SEQ ID NO: 15-19. In some embodiments, the streptavidin-binding peptide includes the sequence of amino acids set forth in SEQ ID NO: 16, also called Twin-Strep-tag®.

II. METHODS FOR PREPARING CHROMATOGRAPHY ARRAYS

[0204] Also provided herein are methods for preparing a chromatography array, including methods for preparing any of the provided chromatography arrays, e.g., any as described in Section I. In some embodiments, the method includes adding a stationary phase to a cavity of each of one or more wells of a spatial array of wells. In some embodiments, the spatial array is any as described in Section I-A. In some embodiments, the stationary phase is any as described in Section I-B. In some embodiments, the method further includes preparing the stationary phase prior to the adding, for instance by immobilizing a selection agent (e.g., any as described in Sections I-B-2 and I-B-3) on a chromatography matrix (e.g., any as described in Section I-B-l), e.g., via a reagent attached to the chromatography matrix (e.g., any as described in Section I-B- 3).

[0205] In some embodiments, the chromatography array is sterile.

[0206] In some embodiments, the selection agent of the added stationary phase is the same across the one or more wells. In some embodiments, the selection agent is different across the one or more wells. In some embodiments, the different selection agents specifically bind to different selection markers, for instance some wells including a first selection agent binding to a first selection marker, and other wells including a second selection agent binding to a second selection marker that is different from the first selection marker. In some embodiments, the different selection agents specifically bind to the same selection marker. As an example, the different selection agents may bind at different epitopes of the selection marker, or may include different formats, such as a monovalent antibody fragment in one set of wells and a divalent antibody fragment in a second set of wells.

[0207] In some embodiments, the amount of stationary phase added to each cavity is to achieve a desired binding capacity for cells expressing the selection marker. In some embodiments, the amount of stationary phase added to each cavity is capable of immobilizing between or between about 0.1 x 10 ⁶ cells and 10 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 9 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 8 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 7 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 6 x 10 ⁶ cells,

0.1 x 10 ⁶ cells and 5 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 4 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 3 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 2 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 1 x 10 ⁶ cells, 0.1 x 10 ⁶ cells and 0.5 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 10 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 9 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 8 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 7 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 6 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and

5 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 4 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 3 x 10 ⁶ cells, 0.5 x 10 ⁶ cells and 2 x 10 ⁶ cells, or 0.5 x 10 ⁶ cells and 1 x 10 ⁶ cells expressing the selection marker, each inclusive. In some embodiments, the amount of stationary phase added to each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells, 1 x 10 ⁶ and 9 x 10 ⁶ cells, 1 x 10 ⁶ and 8 x 10 ⁶ cells, 1 x 10 ⁶ and 7 x 10 ⁶ cells, 1 x 10 ⁶ and 6 x 10 ⁶ cells, 1 x 10 ⁶ and 5 x 10 ⁶ cells, 1 x 10 ⁶ and 4 x 10 ⁶ cells, 1 x 10 ⁶ and 3 x 10 ⁶ cells, 1 x 10 ⁶ and 2 x 10 ⁶ cells, 2 x 10 ⁶ and 10 x 10 ⁶ cells, 2 x 10 ⁶ and 9 x 10 ⁶ cells, 2 x 10 ⁶ and 8 x 10 ⁶ cells, 2 x 10 ⁶ and 7 x 10 ⁶ cells, 2 x 10 ⁶ and 6 x 10 ⁶ cells, 2 x 10 ⁶ and 5 x 10 ⁶ cells, 2 x 10 ⁶ and 4 x 10 ⁶ cells, 2 x 10 ⁶ and 3 x 10 ⁶ cells, 3 x 10 ⁶ and 10 x 10 ⁶ cells, 3 x 10 ⁶ and 9 x 10 ⁶ cells, 3 x 10 ⁶ and 8 x 10 ⁶ cells, 3 x 10 ⁶ and 7 x 10 ⁶ cells, 3 x 10 ⁶ and 6 x 10 ⁶ cells, 3 x 10 ⁶ and 5 x 10 ⁶ cells, 3 x 10 ⁶ and 4 x 10 ⁶ cells, 4 x 10 ⁶ and 10 x 10 ⁶ cells, 4 x 10 ⁶ and 9 x 10 ⁶ cells, 4 x 10 ⁶ and 8 x 10 ⁶ cells, 4 x 10 ⁶ and 7 x 10 ⁶ cells, 4 x 10 ⁶ and 6 x 10 ⁶ cells, 4 x 10 ⁶ and 5 x 10 ⁶ cells, 5 x 10 ⁶ and 10 x 10 ⁶ cells, 5 x 10 ⁶ and 9 x 10 ⁶ cells, 5 x 10 ⁶ and 8 x 10 ⁶ cells, 5 x 10 ⁶ and 7 x 10 ⁶ cells, 5 x 10 ⁶ and 6 x 10 ⁶ cells, 6 x 10 ⁶ and 10 x 10 ⁶ cells, 6 x 10 ⁶ and 9 x 10 ⁶ cells, 6 x 10 ⁶ and 8 x 10 ⁶ cells, 6 x 10 ⁶ and 7 x 10 ⁶ cells, 7 x 10 ⁶ and 10 x 10 ⁶ cells, 7 x 10 ⁶ and 9 x 10 ⁶ cells, 7 x 10 ⁶ and 8 x 10 ⁶ cells, 8 x 10 ⁶ and 10 x 10 ⁶ cells, 8 x 10 ⁶ and 9 x 10 ⁶ cells, or 9 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker, each inclusive. In some embodiments, the amount of stationary phase included in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker.

[0208] In some embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 3000 mg, 10 mg and 2500 mg, 10 mg and 2000 mg, 10 mg and 1500 mg, 10 mg and 1000 mg, 10 mg and 800 mg, 10 mg and 600 mg, 10 mg and 400 mg, 10 mg and 200 mg, 10 mg and 100 mg, 100 mg and 3000 mg, 100 mg and 2500 mg, 100 mg and 2000 mg, 100 mg and 1500 mg, 100 mg and 1000 mg, 100 mg and 800 mg, 100 mg and 600 mg, 100 mg and 400 mg, 100 mg and 200 mg, 200 mg and 3000 mg, 200 mg and 2500 mg, 200 mg and 2000 mg, 200 mg and 1500 mg, 200 mg and 1000 mg, 200 mg and 800 mg, 200 mg and 600 mg, 200 mg and 400 mg, 400 mg and 3000 mg, 400 mg and 2500 mg, 400 mg and 2000 mg, 400 mg and 1500 mg, 400 mg and 1000 mg, 400 mg and 800 mg, 400 mg and 600 mg, 600 mg and 3000 mg, 600 mg and 2500 mg, 600 mg and 2000 mg, 600 mg and 1500 mg, 600 mg and 1000 mg, 600 mg and 800 mg, 800 mg and 3000 mg, 800 mg and 2500 mg, 800 mg and 2000 mg, 800 mg and 1500 mg, 800 mg and 1000 mg, 1000 mg and 3000 mg, 1000 mg and 2500 mg, 1000 mg and 2000 mg, 1000 mg and 1500 mg, 1500 mg and 3000 mg, 1500 mg and 2500 mg, 1500 mg and 2000 mg, 2000 mg and 3000 mg, 2000 mg and 2500 mg, 2500 mg and 3000 mg, each inclusive. In some embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 3000 mg, inclusive. In some embodiments, the amount of stationary phase added to each cavity is between or between about 10 mg and 1000 mg, inclusive. In some embodiments, the amount of stationary phase added to each cavity is between or between about 100 mg and 500 mg, inclusive. In some embodiments, the amount of stationary phase added to each cavity is between or between about 200 mg and 400 mg, inclusive.

[0209] In some embodiments, the method further includes centrifuging the chromatography following the adding. In some embodiments, the stationary phase is added in multiple steps. In some embodiments, the steps of adding and centrifuging are repeated a plurality of times. In some embodiments, the chromatography array is centrifuged after some or all of the multiple steps of adding the stationary phase.

[0210] In some embodiments, the spatial array, e.g., multi-well plate, has been sterilized. In some embodiments, the method further includes sterilizing the spatial array, e.g., multi-well plate. In some embodiments, the spatial array, e.g., multi-well plate, is made of material that can be sterilized. In some embodiments, the sterilizing is performed prior to the adding of the stationary phase. In some embodiments, the spatial array, e.g., multi-well plate, is maintained under sterile conditions until the adding of the stationary phase.

[0211] In some embodiments, the sterilizing is by methods involving wet heat (e.g., autoclaving), dry heat (e.g., flaming or baking), the use of solvents (e.g., disinfectants, such as alcohol, e.g., ethanol or isopropanol), radiation (e.g., UV, x-ray, or gamma irradiation), and/or gas sterilization (e.g., with ethylene oxide). In some embodiments, the spatial array, e.g., multiwell plate, has been autoclaved. In some embodiments, the sterilizing involves autoclaving the spatial array, e.g., multi-well plate. In some embodiments, the spatial array, e.g., multi-well plate, has been gamma irradiated. In some embodiments, the sterilizing involves gamma irradiating the spatial array, e.g., multi-well plate.

III. KITS FOR PREPARING CHROMATOGRAPHY ARRAYS

[0212] Also provided are kits for preparing a chromatography array, including for preparing any of the provided chromatography arrays, e.g., any as described in Section I. In some embodiments, the kit includes instructions for preparing the chromatography array, including according to any of the provided methods, e.g., any as described in Section II. In some embodiments, the kit includes instructions for using the chromatography array, including according to any of the provided methods, e.g., any as described in Section IV.

[0213] In some embodiments, the kit includes a spatial array of wells, e.g., any as described in Section I-A. In some embodiments, the kit includes a chromatography matrix, e.g., any as described in Section I-B-l. In some embodiments, the kit includes a selection agent, e.g., any as described in Sections I-B-2 and I-B-3. In some embodiments, the kit includes instructions for preparing the stationary phase from the chromatography matrix and selection agent, e.g., according to any of the provided methods. In some embodiments, the kit includes a stationary phase in which a selection agent is already immobilized on a chromatography matrix, e.g., any as described in Section I-B.

[0214] In some embodiments, the articles of manufacture or kits include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and/or packaging, generally including instructions for use and instructions to carry out any of the methods provided herein. The kits and articles of manufacture provided herein can contain packaging materials. Packaging materials for use in packaging the provided materials are well known to those of skill in the art. See, for example, U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies, e.g., pipette tips and/or plastic plates, or bottles. The articles of manufacture or kits can include a device so as to facilitate dispensing of the materials or to facilitate use in a high-throughput or large-scale manner, e.g., to facilitate use in robotic equipment. Typically, the packaging is non- reactive with the compositions contained therein.

[0215] In some embodiments, the reagents are packaged separately. In some embodiments, each container can have a single compartment. In some embodiments, other components of the articles of manufacture or kits are packaged separately, or together in a single compartment.

IV. METHODS FOR CELL SELECTION USING CHROMATOGRAPHY ARRAYS

[0216] Also provided herein are methods of using the provided chromatography arrays. In some embodiments, the method is for isolating a target cell. In some embodiments, the target cell is isolated based on its expression or lack of expression of a particular selection marker, e.g., a particular cell surface marker. In some embodiments, the method is for selecting, e.g., positively or negatively selecting, a target cell. In some embodiments, the target cell is selected, e.g., positively or negatively selected, based on its expression or lack of expression of the selection marker. In some embodiments, the target cell expresses the selection marker (positive selection). In some embodiments, the target cell does not express the selection marker (negative selection). In some embodiments, the method is a high-throughput method. In some embodiments, the chromatography array is any as described in Section I.

[0217] In some embodiments, the target cell expresses the selection marker. In some embodiments, the collecting involves binding of the selection agent to the selection marker expressed by the target cell. In some embodiments, the collecting involves flow-through of nontarget cells not expressing the selection marker through the barrier. In some embodiments, the collecting involves flow-through of the target cell through the barrier.

[0218] In some embodiments, the collecting involves adding biotin or a biotin analog to the stationary phase. In some embodiments, the adding of biotin or the biotin analog is prior to the flow-through of the target cell through the barrier. [0219] In some embodiments, the target cell expresses the selection marker, and the collecting involves: binding of the selection agent to the selection marker expressed by the target cell; flow-through of non-target cells not expressing the selection marker through the barrier; and flow-through of the target cell through the barrier. In some embodiments, the collecting involves adding biotin or a biotin analog to the stationary phase prior to the flow-through of the target cell through the barrier. In some embodiments, the target cell expresses the selection marker, and the collecting involves: binding of the selection agent to the selection marker expressed by the target cell; flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and flow-through of the target cell through the barrier. In some embodiments, the target cell expresses the selection marker, and the collecting involves, in order: binding of the selection agent to the selection marker expressed by the target cell; flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and flowthrough of the target cell through the barrier.

[0220] In some embodiments, the collecting involves adding biotin to the stationary phase. In some embodiments, the collecting involves adding D-biotin to the stationary phase. In some embodiments, the collecting involves adding a biotin analog to the stationary phase. In some embodiments, the biotin analog is iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, hydroxyazobenzene-benzoic acid (HABA), or dimethyl-HABA.

[0221] In some embodiments, the biotin or biotin analog disrupts the immobilization of the selection agent to the chromatography matrix. In some embodiments, the biotin or biotin analog disrupts the binding of the binding partner, e.g., streptavidin-binding peptide, of the selection agent to the protein reagent attached to the chromatography matrix. In some embodiments, the biotin or biotin analog disrupts the binding of the binding partner, e.g., streptavidin-binding peptide, of the selection agent to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture of any of the foregoing of the protein reagent. In some embodiments, the biotin or biotin analog disrupts the binding of the binding partner, e.g., streptavidin-binding peptide, of the selection agent to the streptavidin or streptavidin mutein of the protein reagent. In some embodiments, the biotin or biotin analog disrupts the binding of the binding partner, e.g., streptavidin-binding peptide, of the selection agent to the streptavidin mutein of the protein reagent. [0222] In some embodiments, the target cell does not express the selection marker. In some embodiments, the collecting involves binding of the selection agent to non-target cells expressing the selection marker. In some embodiments, the collecting involves flow-through of the target cell through the barrier.

[0223] In some embodiments, the target cell does not express the selection marker, and the collecting involves: binding of the selection agent to non-target cells expressing the selection marker; and flow-through of the target cell through the barrier. In some embodiments, the target cell does not express the selection marker, and the collecting involves, in order: binding of the selection agent to non-target cells expressing the selection marker; and flow-through of the target cell through the barrier.

[0224] In some embodiments, the method involves collecting an output composition of eluted cells. In some embodiments, the eluted cells have passed through the barrier.

[0225] In some embodiments, cells of the output composition have a lower proportion of cells that express the selection marker on their surface compared to the cells of the input composition.

[0226] In some embodiments, the output composition of eluted cells contains at least 30% of the cells of the input composition. In some embodiments, the output composition of eluted cells contains 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 90%, 70% to 80%, or 80% to 90% of the cells of the input composition.

[0227] In some embodiments, the output composition of eluted cells contains 30% to 80% of the cells of the input composition.

[0228] In some embodiments, less than 40% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 35% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 30% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 25% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 20% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 15% of the cells of the output composition of eluted cells express the selection marker on their surface. In some embodiments, less than 10% of the cells of the output composition of eluted cells express the selection marker on their surface.

[0229] Suitable selection markers for the isolation of cells can be chosen based on the cells being targeted. In some embodiments, the selection marker is any as described in Section I-B-2. In some embodiments, the target cell is an immune cell. In some embodiments, the target cell is a lymphocyte. In some embodiments, the target cell is a T cell, B cell, or NK cell. In some embodiments, the target cell is a T cell. Examples of T cells include cells such as CMV-specific CD8+ T cells, 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. In some embodiments, the selection marker is a marker expressed on the surface of T cells. In some embodiments, the selection marker is a T cell coreceptor. In some embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, and/or CD45RO.

[0230] In some embodiments, the method includes adding an input composition including a plurality of cells to the stationary phase of a well of the one or more wells of the chromatography array. In some embodiments, an input composition including a plurality of cells is added to the stationary phase of at least 4, 6, 8, 10, 12, 24, 48, 96, 144, 192, 240, 288, 336, 384, 432, 480, 576, 672, 768, 864, 960, 1056, 1152, 1248, 1344, 1440, or 1536 wells. In some embodiments, the plurality of cells includes one or more cells expressing the selection marker. In some embodiments, the one or more cells expressing the selection marker are immobilized on the stationary phase via binding of the selection marker to the selection agent.

[0231] In some embodiments, following the adding, the method further includes collecting the target cell from the chromatography array. In some embodiments, the collecting includes flow-through of the target cell through the barrier of the chromatography array. In some embodiments, the collecting includes applying a vacuum to the chromatography array. In some embodiments, the collecting includes centrifuging the chromatography array. In some embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, 50 x g and 1800 x g, 50 x g and 1600 x g, 50 x g and 1400 x g, 50 x g and 1200 x g, 50 x g and 1000 x g, 50 x g and 800 x g, 50 x g and 600 x g, 50 x g and 500 x g, 50 x g and 400 x g, 50 x g and 300 x g, 50 x g and 200 x g, 100 x g and 2000 x g, 100 x g and 1800 x g, 100 x g and 1600 x g, 100 x g and 1400 x g, 100 x g and 1200 x g, 100 x g and 1000 x g, 100 x g and 800 x g, 100 x g and 600 x g, 100 x g and 500 x g, 100 x g and 400 x g, 100 x g and 300 x g, 100 x g and 200 x g, 200 x g and 2000 x g, 200 x g and 1800 x g, 200 x g and 1600 x g, 200 x g and 1400 x g, 200 x g and 1200 x g, 200 x g and 1000 x g, 200 x g and 800 x g, 200 x g and 600 x g, 200 x g and 500 x g, 200 x g and 400 x g, 200 x g and 300 x g, 300 x g and 2000 x g, 300 x g and 1800 x g, 300 x g and 1600 x g, 300 x g and 1400 x g, 300 x g and 1200 x g, 300 x g and 1000 x g, 300 x g and 800 x g, 300 x g and 600 x g, 300 x g and 500 x g, 300 x g and 400 x g, 400 x g and 2000 x g, 400 x g and 1800 x g, 400 x g and 1600 x g, 400 x g and 1400 x g, 400 x g and 1200 x g, 400 x g and 1000 x g, 400 x g and 800 x g, 400 x g and 600 x g, 400 x g and 500 x g, 500 x g and 2000 x g, 500 x g and 1800 x g, 500 x g and 1600 x g, 500 x g and 1400 x g, 500 x g and 1200 x g, 500 x g and 1000 x g, 500 x g and 800 x g, 500 x g and 600 x g, 600 x g and 2000 x g, 600 x g and 1800 x g, 600 x g and 1600 x g, 600 x g and 1400 x g, 600 x g and 1200 x g, 600 x g and 1000 x g, 600 x g and 800 x g, 800 x g and 2000 x g, 800 x g and 1800 x g, 800 x g and 1600 x g, 800 x g and 1400 x g, 800 x g and 1200 x g, 800 x g and 1000 x g, 1000 x g and 2000 x g, 1000 x g and 1800 x g, 1000 x g and 1600 x g, 1000 x g and 1400 x g, 1000 x g and 1200 x g,

1200 x g and 2000 x g, 1200 x g and 1800 x g, 1200 x g and 1600 x g, 1200 x g and 1400 x g,

1400 x g and 2000 x g, 1400 x g and 1800 x g, 1400 x g and 1600 x g, 1600 x g and 2000 x g,

1600 x g and 1800 x g, or 1800 x g and 2000 x g, each inclusive. In some embodiments, the centrifuging is performed at between or between about 50 x g and 2000 x g, inclusive. In some embodiments, the centrifuging is performed at between or between about 100 x g and 1000 x g, inclusive. In some embodiments, the centrifuging is performed at between or between about 300 x g and 500 x g, inclusive. In some embodiments, the centrifuging is performed at between or between about 350 x g and 450 x g, inclusive.

[0232] In some embodiments, the centrifuging is performed at between or between about 200 x g and 2000 x g, inclusive. In some embodiments, the centrifuging is performed at between or between about 200 x g and 1500 x g, inclusive.

[0233] In some embodiments, the collecting includes centrifuging the chromatography array. In some embodiments, the centrifuging is performed for between or between about 30 seconds and 10 minutes, 30 seconds and 9 minutes, 30 seconds and 8 minutes, 30 seconds and 7 minutes, 30 seconds and 6 minutes, 30 seconds and 5 minutes, 30 seconds and 4 minutes, 30 seconds and 3 minutes, 30 seconds and 2 minutes, 30 seconds and 1 minute, 1 minute and 10 minutes, 1 minute and 9 minutes, 1 minute and 8 minutes, 1 minute and 7 minutes, 1 minute and 6 minutes, 1 minute and 5 minutes, 1 minute and 4 minutes, 1 minute and 3 minutes, 1 minute and 2 minutes, 2 minutes and 10 minutes, 2 minutes and 9 minutes, 2 minutes and 8 minutes, 2 minutes and 7 minutes, 2 minutes and 6 minutes, 2 minutes and 5 minutes, 2 minutes and 4 minutes, 2 minutes and 3 minutes, 3 minutes and 10 minutes, 3 minutes and 9 minutes, 3 minutes and 8 minutes, 3 minutes and 7 minutes, 3 minutes and 6 minutes, 3 minutes and 5 minutes, 3 minutes and 4 minutes, 4 minutes and 10 minutes, 4 minutes and 9 minutes, 4 minutes and 8 minutes, 4 minutes and 7 minutes, 4 minutes and 6 minutes, 4 minutes and 5 minutes, 5 minutes and 10 minutes, 5 minutes and 9 minutes, 5 minutes and 8 minutes, 5 minutes and 7 minutes, 5 minutes and 6 minutes, 6 minutes and 10 minutes, 6 minutes and 9 minutes, 6 minutes and 8 minutes, 6 minutes and 7 minutes, 7 minutes and 10 minutes, 7 minutes and 9 minutes, 7 minutes and 8 minutes, 8 minutes and 10 minutes, 8 minutes and 9 minutes, or 9 minutes and 10 minutes, each inclusive. In some embodiments, the centrifuging is performed for between or between about 30 seconds and 10 minutes, inclusive. In some embodiments, the centrifuging is performed for between or between about 30 seconds and 5 minutes, inclusive. In some embodiments, the centrifuging is performed for between or between about 2 minutes and 8 minutes, inclusive. In some embodiments, the centrifuging is performed for between or between about 4 minutes and 6 minutes, inclusive.

[0234] In some embodiments, the centrifuging is performed at between or between about 200 x g and 2000 x g, inclusive, for between or between about 30 seconds and 5 minutes, inclusive. In some embodiments, the centrifuging is performed at between or between about 200 x g and 1500 x g, inclusive, for between or between about 30 seconds and 5 minutes, inclusive. In some embodiments, the centrifuging is performed at between or between about 300 x g and 500 x g, inclusive, for between or between about 30 seconds and 5 minutes, inclusive. In some embodiments, the centrifuging is performed at between or between about 300 x g and 500 x g, inclusive, for between or between about 4 minutes and 6 minutes, inclusive.

[0235] In some embodiments, the target cell expresses the selection marker. In some embodiments, the collecting involves binding of the selection agent to the selection marker expressed by the target cell. In some embodiments, the collecting involves centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier. In some embodiments, the collecting involves centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0236] In some embodiments, the collecting involves adding biotin or a biotin analog to the stationary phase. In some embodiments, the adding of biotin or the biotin analog is prior to the flow-through of the target cell through the barrier.

[0237] In some embodiments, the target cell expresses the selection marker, and the collecting involves: binding of the selection agent to the selection marker expressed by the target cell; centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier; and centrifuging the chromatography array for flowthrough of the target cell through the barrier. In some embodiments, the collecting involves adding biotin or a biotin analog to the stationary phase prior to the flow-through of the target cell through the barrier. In some embodiments, the target cell expresses the selection marker, and the collecting involves: binding of the selection agent to the selection marker expressed by the target cell; centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and centrifuging the chromatography array for flow-through of the target cell through the barrier. In some embodiments, the target cell expresses the selection marker, and the collecting involves, in order: binding of the selection agent to the selection marker expressed by the target cell; centrifuging the chromatography array for flow-through of non-target cells not expressing the selection marker through the barrier; adding biotin or a biotin analog to the stationary phase; and centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0238] In some embodiments, the target cell does not express the selection marker. In some embodiments, the collecting involves binding of the selection agent to non-target cells expressing the selection marker. In some embodiments, the collecting involves centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0239] In some embodiments, the target cell does not express the selection marker, and the collecting involves: binding of the selection agent to non-target cells expressing the selection marker; and centrifuging the chromatography array for flow-through of the target cell through the barrier. In some embodiments, the target cell does not express the selection marker, and the collecting involves, in order: binding of the selection agent to non-target cells expressing the selection marker; and centrifuging the chromatography array for flow-through of the target cell through the barrier.

[0240] In some embodiments, the collecting includes applying a vacuum to the chromatography array. In some embodiments, the vacuum is applied for between or between about 30 seconds and 10 minutes, 30 seconds and 9 minutes, 30 seconds and 8 minutes, 30 seconds and 7 minutes, 30 seconds and 6 minutes, 30 seconds and 5 minutes, 30 seconds and 4 minutes, 30 seconds and 3 minutes, 30 seconds and 2 minutes, 30 seconds and 1 minute, 1 minute and 10 minutes, 1 minute and 9 minutes, 1 minute and 8 minutes, 1 minute and 7 minutes, 1 minute and 6 minutes, 1 minute and 5 minutes, 1 minute and 4 minutes, 1 minute and 3 minutes, 1 minute and 2 minutes, 2 minutes and 10 minutes, 2 minutes and 9 minutes, 2 minutes and 8 minutes, 2 minutes and 7 minutes, 2 minutes and 6 minutes, 2 minutes and 5 minutes, 2 minutes and 4 minutes, 2 minutes and 3 minutes, 3 minutes and 10 minutes, 3 minutes and 9 minutes, 3 minutes and 8 minutes, 3 minutes and 7 minutes, 3 minutes and 6 minutes, 3 minutes and 5 minutes, 3 minutes and 4 minutes, 4 minutes and 10 minutes, 4 minutes and 9 minutes, 4 minutes and 8 minutes, 4 minutes and 7 minutes, 4 minutes and 6 minutes, 4 minutes and 5 minutes, 5 minutes and 10 minutes, 5 minutes and 9 minutes, 5 minutes and 8 minutes, 5 minutes and 7 minutes, 5 minutes and 6 minutes, 6 minutes and 10 minutes, 6 minutes and 9 minutes, 6 minutes and 8 minutes, 6 minutes and 7 minutes, 7 minutes and 10 minutes, 7 minutes and 9 minutes, 7 minutes and 8 minutes, 8 minutes and 10 minutes, 8 minutes and 9 minutes, or 9 minutes and 10 minutes, each inclusive. In some embodiments, the vacuum is applied for between or between about 30 seconds and 10 minutes, inclusive. In some embodiments, the vacuum is applied for between or between about 30 seconds and 5 minutes, inclusive. In some embodiments, the vacuum is applied for between or between about 2 minutes and 8 minutes, inclusive. In some embodiments, the vacuum is applied for between or between about 4 minutes and 6 minutes, inclusive.

[0241] In some embodiments, the input composition is from a sample of cells. In some embodiments, cells of the same sample are added to the stationary phase of multiple wells of the chromatography array. In some embodiments, cells of different samples are added to different wells of the chromatography array. In any of these embodiments, the same or different selection agents can be used to bind to the selection marker, including to the same or different selection markers across wells.

[0242] In some embodiments, the input composition includes between or between about 1 x 10 ⁶ cells and 60 x 10 ⁶ cells, 1 x 10 ⁶ cells and 50 x 10 ⁶ cells, 1 x 10 ⁶ cells and 40 x 10 ⁶ cells, 1 x

10 ⁶ cells and 30 x 10 ⁶ cells, 1 x 10 ⁶ cells and 20 x 10 ⁶ cells, 1 x 10 ⁶ cells and 10 x 10 ⁶ cells, 1 x

10 ⁶ cells and 5 x 10 ⁶ cells, 5 x 10 ⁶ cells and 60 x 10 ⁶ cells, 5 x 10 ⁶ cells and 50 x 10 ⁶ cells, 5 x 10 ⁶ cells and 40 x 10 ⁶ cells, 5 x 10 ⁶ cells and 30 x 10 ⁶ cells, 5 x 10 ⁶ cells and 20 x 10 ⁶ cells, 5 x

10 ⁶ cells and 10 x 10 ⁶ cells, 10 x 10 ⁶ cells and 60 x 10 ⁶ cells, 10 x 10 ⁶ cells and 50 x 10 ⁶ cells,

10 x 10 ⁶ cells and 40 x 10 ⁶ cells, 10 x 10 ⁶ cells and 30 x 10 ⁶ cells, 10 x 10 ⁶ cells and 20 x 10 ⁶ cells, 20 x 10 ⁶ cells and 60 x 10 ⁶ cells, 20 x 10 ⁶ cells and 50 x 10 ⁶ cells, 20 x 10 ⁶ cells and 40 x 10 ⁶ cells, 20 x 10 ⁶ cells and 30 x 10 ⁶ cells, 30 x 10 ⁶ cells and 60 x 10 ⁶ cells, 30 x 10 ⁶ cells and 50 x 10 ⁶ cells, 30 x 10 ⁶ cells and 40 x 10 ⁶ cells, 40 x 10 ⁶ cells and 60 x 10 ⁶ cells, 40 x 10 ⁶ cells and 50 x 10 ⁶ cells, or 50 x 10 ⁶ cells and 60 x 10 ⁶ cells, each inclusive. In some of any embodiments, the input composition includes between or between about 10 x 10 ⁶ cells and 50 x 10 ⁶ cells. These input cell numbers can be total cells of the input composition, or they can be the number of cells expressing or expected to express the selection marker. In some embodiments, these input cell numbers are with reference to total cells of the input composition.

[0243] In some of any embodiments, the input composition includes between or between about 1 x 10 ⁶ cells and 50 x 10 ⁶ cells. In some of any embodiments, the input composition includes between or between about 1 x 10 ⁶ cells and 40 x 10 ⁶ cells. These input cell numbers can be total cells of the input composition, or they can be the number of cells expressing or expected to express the selection marker. In some embodiments, these input cell numbers are with reference to total cells of the input composition.

[0244] In some embodiments, the input composition has a cell density of between or between about 5 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 5 x 10 ⁶ cells/mL and 40 x 10 ⁶ cells/mL, 5 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, 5 x 10 ⁶ cells/mL and 20 x 10 ⁶ cells/mL, 5 x 10 ⁶ cells/mL and 10 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 40 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 20 x 10 ⁶ cells/mL, 20 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 20 x 10 ⁶ cells/mL and 40 x 10 ⁶ cells/mL, 20 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, 30 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 30 x 10 ⁶ cells/mL and 40 x 10 ⁶ cells/mL, or 40 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, each inclusive. In some embodiments, the input composition has a cell density of between or between about 5 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, inclusive. In some embodiments, the input composition has a cell density of between or between about 10 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, inclusive. In some embodiments, the input composition has a cell density of between or between about 15 x 10 ⁶ cells/mL and 25 x 10 ⁶ cells/mL, inclusive. These input cell densities can be with reference to total cells of the input composition, or they can be with reference to the number of cells expressing or expected to express the selection marker. In some embodiments, these input cell densities are with reference to total cells of the input composition.

[0245] In some embodiments, the number of cells added to the stationary phase of a well is based on the percentage of added cells expected to express the selection marker. Thus, in some embodiments, the number of cells added to the stationary phase is based on the number of cells of the plurality of cells of the input composition that express the selection marker. In some embodiments, the number of cells added to the stationary phase is based on the binding capacity of the stationary phase for binding to cells expressing the selection marker. In some embodiments, the number of cells added to the stationary phase is based on the amount of stationary phase present in the well. In some embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase included in the cavity of the well is between or between about 1 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 40 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 30 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 5 x 10 ⁶ cells/g, 5 x

10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 5 x 10 ⁶ cells/g and 40 x 10 ⁶ cells/g, 5 x 10 ⁶ cells/g and 30 x 10 ⁶ cells/g, 5 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, 5 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, 10 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 10 x 10 ⁶ cells/g and 40 x 10 ⁶ cells/g, 10 x 10 ⁶ cells/g and 30 x 10 ⁶ cells/g, 10 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, 20 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 20 x 10 ⁶ cells/g and 40 x 10 ⁶ cells/g, 20 x 10 ⁶ cells/g and 30 x 10 ⁶ cells/g, 30 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 30 x 10 ⁶ cells/g and 40 x 10 ⁶ cells/g, or 40 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, each inclusive. In some embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase in the cavity is between or between about 1 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, inclusive. In some embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase in the cavity is between or between about 1 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, inclusive. In some embodiments, the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase in the cavity is between or between about 1 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, inclusive.

[0246] In some embodiments, the method further includes incubating the input composition in the well prior to the collecting of the target cell. In some embodiments, the incubating is to allow for binding of the selection marker to the selection agent and the immobilization of the plurality of cells on the stationary phase. In some embodiments, the incubating is performed for between or between about 2 minutes and 120 minutes, 2 minutes and 110 minutes, 2 minutes and 100 minutes, 2 minutes and 90 minutes, 2 minutes and 80 minutes, 2 minutes and 70 minutes, 2 minutes and 60 minutes, 2 minutes and 50 minutes, 2 minutes and 40 minutes, 2 minutes and 30 minutes, 2 minutes and 20 minutes, 5 minutes and 120 minutes, 5 minutes and 110 minutes, 5 minutes and 100 minutes, 5 minutes and 90 minutes, 5 minutes and 80 minutes, 5 minutes and 70 minutes, 5 minutes and 60 minutes, 5 minutes and 50 minutes, 5 minutes and 40 minutes, 5 minutes and 30 minutes, 5 minutes and 20 minutes, 10 minutes and 120 minutes, 10 minutes and 110 minutes, 10 minutes and 100 minutes, 10 minutes and 90 minutes, 10 minutes and 80 minutes, 10 minutes and 70 minutes, 10 minutes and 60 minutes, 10 minutes and 50 minutes, 10 minutes and 40 minutes, 10 minutes and 30 minutes, 10 minutes and 20 minutes, 20 minutes and 120 minutes, 20 minutes and 110 minutes, 20 minutes and 100 minutes, 20 minutes and 90 minutes, 20 minutes and 80 minutes, 20 minutes and 70 minutes, 20 minutes and 60 minutes, 20 minutes and 50 minutes, 20 minutes and 40 minutes, 20 minutes and 30 minutes, 30 minutes and 120 minutes, 30 minutes and 110 minutes, 30 minutes and 100 minutes, 30 minutes and 90 minutes, 30 minutes and 80 minutes, 30 minutes and 70 minutes, 30 minutes and 60 minutes, 30 minutes and 50 minutes, 30 minutes and 40 minutes, 40 minutes and 120 minutes, 40 minutes and 110 minutes, 40 minutes and 100 minutes, 40 minutes and 90 minutes, 40 minutes and 80 minutes, 40 minutes and 70 minutes, 40 minutes and 60 minutes, 40 minutes and 50 minutes, 50 minutes and 120 minutes, 50 minutes and 110 minutes, 50 minutes and 100 minutes, 50 minutes and 90 minutes, 50 minutes and 80 minutes, 50 minutes and 70 minutes, 50 minutes and 60 minutes, 60 minutes and 120 minutes, 60 minutes and 110 minutes, 60 minutes and 100 minutes, 60 minutes and 90 minutes, 60 minutes and 80 minutes, 60 minutes and 70 minutes, 70 minutes and 120 minutes, 70 minutes and 110 minutes, 70 minutes and 100 minutes, 70 minutes and 90 minutes, 70 minutes and 80 minutes, 80 minutes and 120 minutes, 80 minutes and 110 minutes, 80 minutes and 100 minutes, 80 minutes and 90 minutes, 90 minutes and 120 minutes, 90 minutes and 110 minutes, 90 minutes and 100 minutes, 100 minutes and 120 minutes, 100 minutes and 110 minutes, or 110 minutes and 120 minutes, each inclusive. In some embodiments, the incubating is performed for between or between about 2 minutes and 120 minutes, inclusive. In some embodiments, the incubating is performed for between or between about 2 minutes and 90 minutes, inclusive. In some embodiments, the incubating is performed for between or between about 2 minutes and 60 minutes, inclusive. In some embodiments, the incubating is performed for between or between about 2 minutes and 30 minutes, inclusive.

[0247] In some embodiments, all or a portion of the incubating is performed under mixing conditions. In some embodiments, all of the incubating is performed under mixing conditions. In some embodiments, the mixing conditions include shaking the chromatography array at a speed of between or between about 50 revolutions per minute (rpm) and 1000 rpm, 50 rpm and 800 rpm, 50 rpm and 600 rpm, 50 rpm and 400 rpm, 50 rpm and 200 rpm, 50 rpm and 100 rpm, 100 rpm and 1000 rpm, 100 rpm and 800 rpm, 100 rpm and 600 rpm, 100 rpm and 400 rpm, 100 rpm and 200 rpm, 200 rpm and 1000 rpm, 200 rpm and 800 rpm, 200 rpm and 600 rpm, 200 rpm and 400 rpm, 400 rpm and 1000 rpm, 400 rpm and 800 rpm, 400 rpm and 600 rpm, 600 rpm and 1000 rpm, 600 rpm and 800 rpm, or 800 rpm and 1000 rpm, each inclusive. In some embodiments, the mixing conditions include shaking the chromatography array at a speed of between or between about 50 rpm and 1000 rpm, inclusive. In some embodiments, the mixing conditions include shaking the chromatography array at a speed of between or between about 200 rpm and 800 rpm, inclusive. In some embodiments, the mixing conditions include shaking the chromatography array at a speed of between or between about 400 rpm and 600 rpm, inclusive. In some embodiments, the chromatography array is mixed using an orbital shaker.

[0248] In some embodiments, the input composition and the stationary phase are present together in a slurry in the well. In some embodiments, the mixing conditions effect the mixing of the slurry in the well.

V. DEFINITIONS

[0249] Unless defined otherwise, all terms of art, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0250] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of’ aspects and variations.

[0251] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range is specifically disclosed and is encompassed within the claimed subject matter. In addition, it is understood that subranges between each intervening value in that stated range and any other stated or intervening value in that stated range is specifically disclosed and encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0252] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In certain embodiments, “about X” refers to a value of ±25%, ±10%, ±5%, ±2%, ±1%, ±0.1%, or ±0.01% of X.

[0253] As used herein, recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New. Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[0254] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as retroviral, e.g., gammaretroviral and lentiviral vectors.

[0255] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0256] As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. For instance, when referring to a surface marker, the term can refer to the presence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for a cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

[0257] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. For instance, when referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for a cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0258] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0259] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. [0260] Amino acids generally can be grouped according to the following common sidechain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0261] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0262] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0263] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

VI. EXEMPLARY EMBODIMENTS

[0264] Among the provided embodiments are:

1. A chromatography array comprising a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening, wherein: the cavity of each of the one or more wells comprises a stationary phase, the stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell.

2. The chromatography array of embodiment 1, wherein the selection agent is reversibly immobilized to the chromatography matrix.

3. The chromatography array of embodiment 1 or embodiment 2, wherein the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein: the protein reagent is attached to the chromatography matrix and comprises streptavidin, avidin, a streptavidin mutein, an avidin mutein, or a mixture of any of the foregoing; and the selection agent comprises a binding partner that is bound to the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

4. The chromatography array of embodiment 3, wherein the binding partner is bound to the biotin-binding site of the streptavidin, avidin, streptavidin mutein, avidin mutein, or mixture.

5. The chromatography array of any one of embodiments 1-5, wherein the selection agent is immobilized to the chromatography matrix via a protein reagent, wherein: the protein reagent is attached to the chromatography matrix and comprises streptavidin or a streptavidin mutein; and the selection agent comprises a binding partner that is bound to the streptavidin or streptavidin mutein.

6. The chromatography array of embodiment 5, wherein the binding partner is bound to the biotin-binding site of the streptavidin or streptavidin mutein.

7. The chromatography array of any one of embodiments 3-6, wherein the binding partner is reversibly bound to the protein reagent, thereby effecting the reversible immobilization of the selection agent, wherein the binding is capable of being disrupted by the addition of a competition reagent to the stationary phase.

8. The chromatography array of embodiment 7, the competition reagent comprises biotin or a biotin analog.

9. The chromatography array of any one of embodiments 3-8, wherein the binding partner comprises biotin, a biotin analog, or a streptavidin-binding peptide.

10. The chromatography array of any one of embodiments 3-9, wherein the binding partner comprises a streptavidin-binding peptide.

11. The chromatography array of embodiment 9 or embodiment 10, wherein the streptavidin-binding peptide comprises the amino acid sequence Trp-Arg-His-Pro-Gln-Phe-Gly- Gly (SEQ ID NO: 7) or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

12. The chromatography array of any one of embodiments 9-11, wherein the streptavidin-binding peptide comprises two or more streptavidin-binding modules. 13. The chromatography array of embodiment 12, wherein each of the two or more streptavidin-binding modules comprises an amino acid sequence independently selected from Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8).

14. The chromatography array of any one of embodiments 9-13, wherein the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15), SAWSHPQFEK(GGGS) ₂ GGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu- Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu- Lys (SEQ ID NO: 18), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-A la-Trp-Ser-His-Pro-Gln-Phe- Glu-Lys (SEQ ID NO: 19).

15. The chromatography array of any one of embodiments 9-14, wherein the streptavidin-binding peptide comprises the amino acid sequence SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 16).

16. The chromatography array of any one of embodiments 3-15, wherein the streptavidin mutein comprises one or more mutations compared to a minimal streptavidin sequence, wherein: the minimal streptavidin sequence is a fragment of wild-type streptavidin that is shortened at the N- and/or the C-terminus; and the N-terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 10 to 16 of the sequence of amino acids set forth in SEQ ID NO: 1, and the C- terminal amino acid residue of the minimal streptavidin sequence is in the region of positions 133 to 142 of the sequence of amino acids set forth in SEQ ID NO: 1.

17. The chromatography array of any one of embodiments 3-16, wherein the streptavidin mutein comprises the amino acid sequence Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Val^-Thr ⁴⁵ - Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO: 1.

18. The chromatography array of any one of embodiments 3-17, wherein the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, 28, 39, and 40. 19. The chromatography array of any one of embodiments 3-18, wherein the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6.

20. The chromatography array of any one of embodiments 1-19, wherein the selection agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an MHC molecules, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing, that specifically binds to the selection marker.

21. The chromatography array of any one of embodiments 1-20, wherein the selection agent comprises a monovalent antibody fragment that specifically binds to the selection marker.

22. The chromatography array of any one of embodiments 1-21, wherein the selection agent comprises a Fab that specifically binds to the selection marker.

23. The chromatography array of any one of embodiments 1-22, wherein the selection marker is a marker expressed on the surface of T cells.

24. The chromatography array of any one of embodiments 1-23, wherein the chromatography matrix comprises a chromatography resin.

25. The chromatography array of any one of embodiments 1-24, wherein the chromatography matrix comprises a polystyrene resin.

26. The chromatography array of any one of embodiments 1-25, wherein the chromatography matrix comprises chromatography resin beads.

27. The chromatography array of embodiment 26, wherein the chromatography resin beads are between or between about 50 pm and 150 pm, 75 pm and 125 pm, or 90 pm and 110 pm in diameter, each inclusive.

28. The chromatography array of any one of embodiments 1-27, wherein the amount of stationary phase comprised in each cavity is capable of immobilizing between or between about 1 x 10 ⁶ and 10 x 10 ⁶ cells expressing the selection marker.

29. The chromatography array of any one of embodiments 1-28, wherein the amount of stationary phase comprised in each cavity is between or between about 10 mg and 3000 mg, 10 mg and 1000 mg, 100 mg and 500 mg, or 200 mg and 400 mg, each inclusive.

30. The chromatography array of any one of embodiments 1-29, wherein the barrier is positioned at, flush with, and/or secured to the bottom opening. 31. The chromatography array of any one of embodiments 1-29, wherein the barrier is positioned and/or secured within the cavity.

32. The chromatography array of any one of embodiments 1-31, wherein the barrier is permeable to T cells.

33. The chromatography array of any one of embodiments 1-32, wherein the barrier is not permeable to the stationary phase.

34. The chromatography array of any one of embodiments 1-33, wherein the barrier comprises pores that are at least or at least about 10 pm in diameter.

35. The chromatography array of any one of embodiments 1-34, wherein the barrier comprises pores that are between or between about 10 pm and 150 pm, 15 pm and 100 pm, or 20 pm and 50 pm in diameter.

36. The chromatography array of any one of embodiments 1-35, wherein the barrier comprises polypropylene and/or polyethylene media.

37. The chromatography array of any one of embodiments 1-36, wherein the spatial array comprises, comprises about, or comprises at least 12 wells, 24 wells, 48 wells, or 96 wells.

38. The chromatography array of any one of embodiments 1-37, wherein each of the one or more wells comprises a volume of between or between about 0.2 mL and 20 mL, 0.2 mL and 10 mL, or 0.5 mL and 1.5 mL, each inclusive.

39. The chromatography array of any one of embodiments 1-38, wherein the spatial array comprises a multi- well plate.

40. The chromatography array of embodiment 39, wherein the multi-well plate is a multi- well filter plate.

41. A method for preparing the chromatography array of any one of embodiments 1- 40, comprising adding a stationary phase to a cavity of each of one or more wells of a spatial array of wells, wherein: each of the one or more wells comprises (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; and the stationary phase comprises a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell. 42. A method for preparing the chromatography array of any one of embodiments 1- 40, comprising:

(a) obtaining a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; and

(b) adding a stationary phase to the cavity of each of the one or more wells, the stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell.

43. The method of embodiment 41 or embodiment 42, further comprising preparing the stationary phase prior to the adding, the preparing comprising immobilizing the selection agent on the chromatography matrix.

44. The method of any one of embodiments 41-43, further comprising centrifuging the chromatography array following the adding.

45. The method of embodiment 44, wherein the steps of adding and centrifuging are repeated a plurality of times.

46. A kit for preparing the chromatography array of any one of embodiments 1-40, comprising:

(i) a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening;

(ii) a chromatography matrix;

(iii) a selection agent that is capable of being immobilized on the chromatography matrix and specifically binds to a selection marker expressed on the surface of a cell; and

(iv) instructions for preparing the chromatography array.

47. A kit for preparing the chromatography array of any one of embodiments 1-40, comprising:

(i) a spatial array of wells, each of one or more of the wells comprising (i) a cavity comprising a top opening and a bottom opening and (ii) a barrier that is permeable to cells and positioned proximal to the bottom opening; (ii) a stationary phase comprising a chromatography matrix having a selection agent immobilized thereon, wherein the selection agent specifically binds to a selection marker expressed on the surface of a cell; and

(iii) instructions for preparing the chromatography array.

48. A method for isolating a target cell, comprising:

(a) adding an input composition comprising a plurality of cells to the stationary phase of a well of the one or more wells of the chromatography array of any one of embodiments 1-40, the plurality of cells comprising one or more cells expressing the selection marker, thereby immobilizing the one or more cells on the stationary phase via binding of the selection marker to the selection agent; and

(b) collecting a target cell from the chromatography array.

49. A method for isolating a target cell, comprising:

(a) obtaining the chromatography array of any one of embodiments 1-40;

(b) adding an input composition comprising a plurality of cells to the stationary phase of a well of the one or more wells of the chromatography array, the plurality of cells comprising one or more cells expressing the selection marker, thereby immobilizing the one or more cells on the stationary phase via binding of the selection marker to the selection agent; and

(c) collecting a target cell from the chromatography array.

50. The method of embodiment 48 or embodiment 49, wherein the target cell expresses the selection marker.

51. The method of embodiment 48 or embodiment 49, wherein the target cell does not express the selection marker.

52. The method of any one of embodiments 48-51, wherein the selection marker is a marker expressed on the surface of T cells.

53. The method of any one of embodiments 48-52, wherein the target cell is a T cell.

54. The method of any one of embodiments 48-53, wherein the input composition comprises between or between about 10 x 10 ⁶ cells and 50 x 10 ⁶ cells.

55. The method of any one of embodiments 48-54, wherein the input composition comprises a cell density of between or between about 5 x 10 ⁶ cells/mL and 50 x 10 ⁶ cells/mL, 10 x 10 ⁶ cells/mL and 30 x 10 ⁶ cells/mL, or 15 x 10 ⁶ cells/mL and 25 x 10 ⁶ cells/mL, each inclusive. 56. The method of any one of embodiments 48-55, wherein the ratio of (i) the number of cells of the plurality of cells that express the selection marker to (ii) the amount of stationary phase comprised in the cavity is between or between about 1 x 10 ⁶ cells/g and 50 x 10 ⁶ cells/g, 1 x 10 ⁶ cells/g and 20 x 10 ⁶ cells/g, or 1 x 10 ⁶ cells/g and 10 x 10 ⁶ cells/g, each inclusive.

57. The method of any one of embodiments 48-56, further comprising incubating the input composition in the well prior to the collecting.

58. The method of embodiment 57, wherein the incubating is performed for between or between about 2 minutes and 120 minutes, 2 minutes and 90 minutes, 2 minutes and 60 minutes, or 2 minutes and 30 minutes, each inclusive.

59. The method of embodiment 57 or embodiment 58, wherein all or a portion of the incubating is performed under mixing conditions.

60. The method of embodiment 59, wherein the mixing conditions comprise shaking the chromatography array at a speed of between or between about 50 revolutions per minute (rpm) and 1000 rpm, 200 rpm and 800 rpm, or 400 rpm and 600 rpm, each inclusive.

61. The method of any one of embodiments 48-60, wherein the collecting comprises flow-through of the target cell through the barrier.

62. The method of any one of embodiments 48-61, wherein the collecting comprises centrifuging the chromatography array or applying a vacuum to the chromatography array.

63. The method of any one of embodiments 48-62, wherein the collecting comprises centrifuging the chromatography array.

64. The method of embodiment 62 or embodiment 63, wherein the centrifuging is performed at between or between about 50 g and 2000 g, 100 g and 1000 g, 300 g and 500 g, or 350 g and 450 g, each inclusive.

VII. EXAMPLES

[0265] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Development of a High-Throughput Affinity-Resin Cell Isolation Method

[0266] This example describes a high-throughput affinity-resin method for isolation of cells based on cell surface marker expression. The method can be used in scale-down screenings using spatial arrays, such as multi-well plates. [0267] For cell isolation, an anti-CD3 affinity resin was used. To prepare the resin, a multimerized streptavidin mutein (Strep-Tactin® m2, SEQ ID NO: 6) was coupled to an epoxyactivated polystytrene resin (CY17030; Cytosorbents) via solvent-exposed primary amine groups. The coupling reaction was performed in phosphate buffered saline (PBS) and at a 0.005:4 weight ratio of multimerized streptavidin mutein to dry resin. Subsequently, a selection agent that included an anti-CD3 Fab fragment and a streptavidin-binding peptide (Twin-Strep- tag®, SEQ ID NO: 16) carboxyl-terminally fused to the heavy chain of the Fab fragment was added to a 50% suspension (resin bed volume vs. total volume) of the Strep-Tactin®-coated resin. The anti-CD3 Fab fragment was derived from the anti-CD3 monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRE-8001™; see also U.S. Patent No. 4,361,549) and contained the heavy chain variable domain (SEQ ID NO: 31) and the light chain variable domain (SEQ ID NO: 32) of the anti-CD3 antibody OKT3 described in Arakawa et al., J. Biochem. 120, 657-662 (1996). The selection agent was added at a 1:5.56 weight ratio of selection agent to multimerized streptavidin mutein, and the suspension was incubated under gentle shaking for one hour at room temperature in order to reversibly bind the Twin-Strep-tag® to the immobilized Strep-Tactin® multimer.

[0268] Next, wells of an autoclaved 96-well filter plate (e.g., Pall Corporation; 1 mF volume per well; non-woven polypropylene/polyethylene filter media; 30-40 pm pore size) were filled with 50 pL or 200 pL of buffer solution containing the anti-CD3 resin at a ratio of 118 mg of resin to 200 pL of buffer solution. The filter plate was centrifuged to elute the buffer solution, then wells were rinsed with additional buffer.

[0269] Following filter plate preparation, 800 pL of an input cell composition containing 35 x 10 ⁶ cells/mL in PlasmaLyte-A solution with 1.25% human serum albumin (HSA) were added per well. Prior to addition, it was estimated that the input composition had 93.5% starting viability and 30.9% CD3 expression. Cells of the input composition were then incubated in the filter plate for 2 minutes or 15 minutes on an orbital plate shaker operating at 500 rpm. Following incubation, the filter plate was centrifuged at 500 x g or 1500 x g for 60 seconds to elute unbound cells into a receiving plate. These negative fractions were then analyzed for cell count, viability, and CD3 expression.

[0270] Results shown in Table El are average values across wells per filter plate. Each test condition was performed on a separate filter plate. As shown in Table El, approximately 45%- 78% of added cells across test conditions were recovered in the negative fraction. In addition, each test condition resulted in negative fractions with a lower percentage of CD3+ cells compared to the input composition, consistent with CD3+ cells of the input composition being bound by the anti-CD3 resin during incubation and remaining bound thereon during centrifugation. As also shown in Table El, viability of the negative fraction was increased compared to that of the input composition.

Table El: Cell Recovery and Viability in Negative Fraction

[0271] Together, these results indicate that the developed affinity-resin method can be used to isolate cells, for instance a negative fraction of cells, with spatial arrays, e.g., filter plates, that can be used in a high-throughput cell isolation method.

Example 2: Effects of Cell Density and Centrifugation Speed on High-Throughput Affinity-Resin Cell Isolation Method

[0272] The effects of varying the cell density of input compositions processed in the high- throughput array-based affinity-resin method described in Example 1 were studied. The effects of varying the speed at which filter plates were centrifuged following incubation were also studied. [0273] In this study, wells of the filter plate were filled with 118 mg of the anti-CD3 resin described in Example 1. Following centrifugation and rinsing, 800 pL of an input cell composition containing 10 x 10 ⁶ cells/mL or 20 x 10 ⁶ cells/mL in PlasmaLyte A solution with 1.25% HSA were added per well. Prior to addition, it was estimated that the input composition was 74% CD3-. Cells of the input composition were then incubated in the filter plate for 15 minutes on an orbital plate shaker operating at 500 rpm. Following incubation, the filter plate was centrifuged at 200 x g, 300 x g, 400 x g, or 500 x g for 5 minutes to elute unbound cells into a receiving plate. These negative fractions were then analyzed for cell count, viability, and CD3 expression.

[0274] As shown in FIG. 1, cell density and centrifugation speed had varying effects on the percentage of viable cells recovered in the negative fractions. In addition, each test condition resulted in negative fractions with a higher percentage of CD3- cells compared to the input composition, consistent with CD3+ cells of the input composition being bound by the anti-CD3 resin during incubation and remaining bound thereon during centrifugation. Based on these findings, it was determined that an input cell density of 20 x 10 ⁶ cells/mE and centrifugation at 400 x g resulted in negative fractions with balanced high purity and yield (condition indicated with a star in FIG. 1). In contrast, an input cell density of 20 x 10 ⁶ cells/mE and centrifugation at 200 x g resulted in negative fractions with high purity, but reduced yield.

[0275] Together, these results indicate that the developed high-throughput array-based affinity-resin method can be used to generate isolated cell compositions with balanced high purity and yield, for instance resulting in isolated cell compositions with purity and cell counts sufficient for further downstream processing and experiments.

Example 3: Effects of Cell-to-Resin Ratio on High-Throughput Affinity-Resin Cell Isolation Method

[0276] The effects of varying the amount of affinity resin used in the high-throughput arraybased method described in Example 1 were studied.

[0277] In this study, an input cell composition estimated to be 81% CD3- was processed. Based on this estimate, the amount of anti-CD3 resin added per well of the filter plate was varied in order to test various ratios of expected CD3+ cells to resin. Following centrifugation and rinsing, 400 pF of the input composition (density 20 x 10 ⁶ cells/mE in PlasmaEyte-A solution with 1.25% HSA) were added per well. Cells of the input composition were then incubated in the filter plate for 15 minutes on an orbital plate shaker operating at 500 rpm. Following incubation, the filter plate was centrifuged at 400 x g for five minutes to elute unbound cells into a receiving plate. These negative fractions were then analyzed for cell count, viability, and CD3 expression.

[0278] As shown in FIG. 2, each test condition resulted in negative fractions with a higher percentage of CD3- cells compared to the input composition. Tested cell-to-resin ratios are shown in units of 1 x 10 ⁶ CD3+ cells/g of resin. As also shown in FIG. 2, lower cell-to-resin ratios resulted in negative fractions with a higher percentage of CD3- cells compared to those achieved with higher cell-to-resin ratios. Further analysis revealed that the lower cell-to-resin ratios used in the high-throughput filter-based affinity-resin method (e.g., the cell-to-resin ratios that achieved the most increase in negative fraction purity) were also lower than the cell-to-resin ratio used in an alternative process for cell isolation using the anti-CD3 resin in a full-scale chromatography column (resin bed volume of 18-20 mL, with up to 2 x 10 ⁹ cells captured per full-scale column).

[0279] Together, these results indicate that cell isolation in the developed high-throughput array-based method can be greater by using higher amounts of affinity resin. These results are consistent with a finding that more resin per cell is needed in the array-based method than in a chromatography column-based isolation method with the same resin.

Example 4: Expansion of Cells Isolated using High-Throughput Affinity-Resin Cell Isolation Method

[0280] Cells isolated using the high-throughput array-based affinity-resin method described in Example 1 were stimulated and expanded. The expansion of cells isolated using this method was compared to that of cells isolated using an alternative, microbead-based isolation method.

[0281] In this study, an anti-CD57 resin was used. The anti-CD57 resin was prepared by adding a selection agent that included an anti-CD57 Fab fragment and a streptavidin-binding peptide (Twin-Strep-tag®, SEQ ID NO: 16) carboxyl-terminally fused to the heavy chain of the Fab fragment to the Strep-Tactin®-coated resin described in Example 1.

[0282] For stimulating cells, an anti-CD3/anti-CD28 stimulatory reagent was prepared using an oligomeric streptavidin mutein reagent produced as described in WO2018/197949 (see also Poltorak et al., Scientific Reports (2020)). The oligomeric streptavidin mutein reagent had an average hydrodynamic radius of 100-140 nm and contained an average of 2000-2800 streptavidin mutein tetramers. The oligomeric streptavidin mutein reagent was mixed at room temperature with the anti-CD3 Fab fragments described in Example 1 as well as with anti-CD28 Fab fragments also individually fused at the carboxy-terminus of their heavy chain to a streptavidin-binding peptide (Twin-Strep-tag®, SEQ ID NO: 16). The anti-CD28 Fab fragments were derived from antibody CD28.3 (deposited as a synthetic single chain Fv construct under GenBank Accession No. AF451974.1; see also Vanhove et al., BEOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570) and contained the heavy chain variable domain (SEQ ID NO: 33) and the light chain variable domain (SEQ ID NO: 34) of the anti-CD28 antibody CD28.3.

[0283] For cell isolation, wells of the filter plate were filled with 118 mg of the anti-CD57 resin. Following centrifugation and rinsing, 800 pF of an input cell composition containing 20 x 10 ⁶ cells/mE in PBS buffer solution with EDTA and 0.5% HSA were added per well. Cells of the input composition were then incubated in the filter plate for 15 minutes on an orbital plate shaker operating at 500 rpm. Following incubation, the filter plate was centrifuged at 400 x g for five minutes to elute unbound cells into a receiving plate. These negative fractions were then analyzed for cell count, viability, and CD57 expression, after which they were then incubated over multiple days in the presence of the anti-CD3/anti-CD28 stimulatory reagent at a concentration of 4 pg of stimulatory reagent per 10 ⁶ cells.

[0284] For comparison, cells of the input composition were isolated using an alternative scale-down method using magnetic CD57 MicroBeads (Miltenyi Biotec). In this process, cells were isolated according to manufacturer instructions, and negative fractions were analyzed and stimulated as described above.

[0285] As shown in FIG. 3A, cell isolation using either the array-based affinity-resin method or the alternative microbead-based method resulted in negative fractions with a higher percentage of CD57- cells compared to cells from the input composition not subjected to a cell isolation method. As shown in FIG. 3B, incubation in the presence of the stimulatory reagent resulted in cell expansion over time. Negative fractions isolated using the array-based affinityresin method achieved greater expansion than did cell compositions not subjected to cell isolation or negative fractions isolated using the microbead-based method.

[0286] Together, these results indicate that cells isolated using the developed array-based affinity-resin method can be stimulated and expanded following cell isolation. In addition, these results indicate that the developed array-based affinity-resin method results in cells with increased capacity for expansion, including compared to cells isolated using an alternative microbead-based method.

Example 5: High-Throughput Affinity-Resin Cell Isolation Method in a 24- Well Plate

[0287] Test conditions for performing the high-throughput affinity-resin method in a 24- well plate were evaluated.

[0288] Wells of an autoclaved 24-well filter plate (e.g., Pall Corporation; 7 mL volume per well; non-woven polypropylene/polyethylene filter media; 30-40 pm pore size) were filled with buffer solution containing the anti-CD3 resin described in Example 1. The anti-CD3 resin was hydrated in IX DPBS + 0.5% HSA. Cell-to-resin ratios of 5 x 10 ⁶ CD3+ cells/g of resin and 20 x 10 ⁶ CD3+ cells/g of resin were tested. The amount of resin to add to each well was determined based on the input cell composition, which was estimated to be 75% CD3- (afterwards determined via flow cytometry to be 73.6% CD3-), and a set volume of 1 mL of the input composition (density 20 x 10 ⁶ cells/mL) to be added per well. Following the addition of resin to reach the cell-to-resin ratio targets, 3 mL of buffer (IX DPBS + 0.5% HSA) was added to each well, and the filter plate was centrifuged at 1500 x g for 60 seconds. The resin was added to the wells in liquid suspension.

[0289] Following filter plate preparation, 1 mL of the input composition (density 20 x 10 ⁶ cells/mL in PlasmaLyte-A solution with 1.25% HSA) was added per well. Cells of the input composition were then incubated in the filter plate for 15 minutes on an orbital plate shaker operating at 300 rpm or 500 rpm. Following incubation, the filter plate was centrifuged at 250 x g or 400 x g for 5 minutes to elute unbound cells into a receiving plate. These negative fractions were then analyzed for cell count, viability, and CD3 expression. Results shown in Table E2 are average values across wells. For comparison, a 96-well filter plate was used in which 400 pL of the input composition (density 20 x 10 ⁶ cells/mL in PlasmaLyte-A solution with 1.25% HSA) was added per well, and in which a cell-to-resin ratio of 5 x 10 ⁶ CD3+ cells/g of resin, a shaker speed of 500 rpm, and a centrifugation speed of 400 x g was used.

[0290] As shown in Table E2, cell recovery was higher when a cell-to-resin ratio of 20 x 10 ⁶ CD3+ cells/g of resin was used in the 24-well filter plate. In addition, cell recovery was higher when a shaker speed of 500 rpm was used with the 24-well filter plate. Centrifugation speed did not affect cell recovery when using the 24-well filter plate. All tested conditions resulted in a decrease in viability with the 24-well filter plate. A cell-to-resin ratio of 5 x 10 ⁶ CD3+ cells/g of resin, a shaker speed of 500 rpm, and a centrifugation speed of 400 x g resulted in the best performance with the 24-well filter plate, which were the conditions used for the 96- well filter plate. Compared to the 96-well filter plate, cell recovery was slightly decreased with the 24-well filter plate, but lower CD3 expression in the negative fraction was achieved with the 24-well filter plate.

Table E2: Cell Recovery and CD3 Expression in Negative Fraction

[0291] Together, these results indicate that the developed affinity-resin method can be used to isolate cells using a 24-well filter plate in which wells had an increased volume and radius and a decreased resin bed aspect ratio per well, compared to wells of a 96-well filter plate. Comparable performance was achieved between the 24-well and 96-well filter plates using the same cell-to-resin ratio, shaker speed, and centrifugation speed conditions.

[0292] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Sequences