Isolation of Cells and Other Complex Biological Materials by Chromatography

Abstract

A method for isolating target cells is provided. [Solution] Target cells (2) have receptor molecules (4) on their cell surface. The method includes the steps of: i) providing a sample containing the target cells; ii) providing a receptor-binding reagent (1) containing a binding site B (3) and a binding partner C (5), where the binding partner C (5) in the receptor-binding reagent is capable of reversibly binding to a binding site Z (6) of an affinity reagent (8); and iii) chromatographing the sample on a suitable stationary phase on which the affinity reagent (8) containing the binding site Z (6) is immobilized, where the binding site Z (6) reversibly binds to the binding partner C (5) on the receptor-binding reagent (1), and the binding site B (3) of the receptor-binding reagent (1) binds to the receptor molecule (4) on the surface of the target cells (2), thereby reversibly immobilizing the target cells (2) on the stationary phase.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 602,150, "Chromatographic Isolation Of Cells And Other Complex Biological Materials," filed with the United States Patent and Trademark Office on February 23, 2012, the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Field of the Invention The present invention relates to the chromatographic isolation of target cells or different (complex) biological materials, particularly by column chromatography, such as affinity chromatography or gel permeation chromatography. The present invention uses receptor - binding reagents that bind to receptor molecules located on the surface of target cells. The methods disclosed herein can also be described as (traceless) cell affinity chromatography technology (CATCH). The present invention generally provides novel methods for isolating biological materials, such as cells, organelles, viruses, etc., without leaving a trace. The present invention also relates to an apparatus for isolating cells and other complex biological materials.

Background Art

[0003] Background of the Invention The isolation of a pure and functional cell population of a desired cell type is essential in various therapeutic, diagnostic, and biotechnological applications.

[0004] Bonnafous et al, J. Immunol. Methods. 1983 Mar 11;58(1-2):93-107 (Non-Patent Document 1) describes cell affinity chromatography using a ligand immobilized via a cleavable mercury-sulfur bond, which means a ligand immobilized via a covalent bond. In this method, Bonnafous et al conjugated the organic mercury compound mersalyl to trisacryl beads having primary amino groups. According to Bonnafous et al, the thiolated ligand can be covalently immobilized on this matrix via a cleavable Hg-S bond. Two model studies of cell separation have been reported by Bonnafous et al: (i) Concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacryl were eluted from the support by a brief thiol treatment that maintained cell viability; (ii) anti-dinitrophenyl antibody modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-trisacryl; sheep red blood cells pre-labeled with trinitrobenzenesulfonic acid bound to this support and were recovered by thiol treatment without hemolysis.

[0005] Note that in this context, chromatography is a well-established technique for separating low molecular weight molecules from high molecular weight molecules, including proteins. This technique is also applied to cell separation, particularly in the form of affinity chromatography, which uses immobilized ligands specific to a desired cell type, e.g., an immunoligand. As an example, various T cell subsets have been separated by loading them onto a column containing polyacrylamide beads covalently bound to rabbit anti-mouse IgG, which is labeled with monoclonal immunoglobulins (Braun, R., et al, Journal of Immunological Methods (1982) 54, 251-258 (Non-Patent Document 2)). As a further example, lectin affinity column chromatography using Sepharose 6MB covalently conjugated to horsegram (Dolichos biflorus) lectin has been used to separate leukemia cells from healthy white blood cells (Ohba, H., et al, Cancer Letters (2002) 184, 207-214 (Non-Patent Document 3)).

[0006] Cells are generally larger than proteins, so, in contrast to proteins, cells hardly enter the pores of the beads of conventional chromatographic adsorbents. Even when using adsorbents with large pores, this separation phenomenon is not significantly improved due to diffusion limitations. On the other hand, the internal surface area of pores accessible only to proteins usually greatly exceeds the surface area accessible to both proteins and cells. Therefore, to use conventional chromatographic adsorbents to immobilize proteinaceous or other receptor-binding ligands to generate an affinity matrix for cells, the use of a wasteful large excess of receptor-binding ligands is required. This is because most of it is usually immobilized in pores or voids inaccessible to cells. Specific receptor-binding reagents are often expensive and difficult to produce on the desired scale, making this aspect a serious consideration. Therefore, the use of monolithic adsorbents in the form of cryogels has been proposed as an alternative approach in cell affinity chromatography (see, for example, Dainiak, M.B., et al., Adv. Biochem. Engin. / Biotechnol. (2007), 106, 101-127 (Non-Patent Document 4)). However, since monolithic adsorbents are rare, the desired adsorbent may not be commercially available in the form of a monolithic column. Furthermore, in the case of affinity chromatography, there generally remains a need to remove the competing compounds used to elute the desired cells from these cells. Therefore, the potential advantages of monolithic adsorbents regarding cell viability can be reversed by additional procedures required to remove the compounds used to elute cells from the affinity chromatography column.

[0007] The most important cell isolation methods currently in use are magnet-assisted cell sorting (MACS) and fluorescence-assisted cell sorting (FACS (trademark)). Typically, cell sorting by flow cytometry, in which fluorophores bound to antibodies are used to label cells, analyzes cells individually. The cells are separated at high speed under ultra-high pressure using a cell sorting device. In FACS (trademark) technology, one-step isolation of cells defined by a marker set is made possible by applying the corresponding set of antibodies with different fluorophores. Therefore, this method is reliable but time-consuming, extremely costly, and cumbersome. In particular, in the case of selection from an apheresis product containing a very large and diverse cell population, e.g., 1×10 10 cells, the very long sorting times of the flow cytometer are not acceptable for an appropriate selection process. Another drawback of FACS (trademark) is that it is very difficult to adapt the complex and interference-prone flow cytometer to the GMP environment required for the isolation of therapeutic cell products. Furthermore, the effector function of the cells may be impaired due to the pressure applied during the cell selection procedure.

[0008] Magnetic-assisted isolation of cells is a system widely used for research and therapeutic applications. The yield and purity of the isolated cells are moderate compared to FACS™ technology, but this selection procedure is robust and does not require high automation. The main drawback of magnetic-assisted isolation is that staining reagents, including magnetic beads on the isolated cells, may remain, which can impair the effector function of the isolated cell population. Furthermore, due to the presence of these magnetic reagents on the isolated cells, a continuous positive selection process is not possible. A continuous positive selection procedure is essential for the selection of cell populations defined by a marker set. Streptamer® technology, described, for example, in international patent application WO02 / 054065 (patent document 1) and US patent No. 7,776,562 (patent document 2), significantly improves cell isolation while utilizing magnetic or fluorescent labeling. In this technology, receptor-binding reagents that exhibit low-affinity binding to receptors located on the surface of cells are used for reversible staining and isolation of cells. In contrast to currently used single positive selection (aimed at removing all cell populations except one cell population of interest), which is combined with magnetic negative selection, continuous positive selection using Streptamer® technology, which involves removal of low-affinity receptor-binding reagents after each selection, results in cell populations with very high purity and yield.

[0009] The object of the present invention is to provide a method and apparatus for overcoming the drawbacks of known techniques for isolating cells, such as FACS™ and MACS technology as described. For example, the present invention aims to provide a rapid, efficient, and gentle cell selection procedure that particularly enables continuous positive cell selection for isolating complex cell populations, such as regulatory T cells or central memory T cells, for research purposes, diagnostic purposes, and particularly therapeutic purposes. Ideally, this novel method and apparatus should also be suitable for the isolation of other complex biological materials than cells.

[0010] This object is solved by the subject matter of the independent claims, in particular, by a method, use, and arrangement as recited in the independent claims.

Prior art documents

Patent documents

[0011]

Patent Document 1

Patent Document 2

Non-patent documents

[0012]

Non-patent Document 1

Non-patent Document 2

Non-patent Document 3

Non-patent Document 4

Summary of the invention

[0013] Gist of the invention The present invention provides a method, kit, arrangement, combination of reagents, and use of a chromatography stationary phase for isolating desired cells having known receptor molecules on their surfaces, including the separation of such cells from other cells lacking such receptors on their surfaces.

[0014] According to a first aspect, the present invention provides a method for isolating a target cell, wherein the target cell has a receptor molecule on the target cell surface, and the method comprises: providing a sample containing the target cell; providing a receptor-binding reagent comprising a binding site B and a binding partner C, wherein the binding site B contained in the receptor-binding reagent can specifically bind to the receptor molecule on the target cell surface, and the dissociation constant (K D ) for the binding of the receptor-binding reagent to the receptor molecule via the binding site B is of low affinity, or the dissociation rate constant (koff) for the binding of the receptor-binding reagent to the receptor molecule via the binding site B has a value of about 3×10 -5 sec -1 or greater, and the binding partner C contained in the receptor-binding reagent can reversibly bind to the binding site Z of the affinity reagent; and subjecting the sample to chromatography on a suitable stationary phase, wherein an affinity reagent is immobilized on the stationary phase, the affinity reagent comprises a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor-binding reagent, and the binding site B of the receptor-binding reagent binds to the receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase; which comprises.

[0015] According to a second aspect, the present invention provides a method for isolating a target cell, wherein the target cell has a receptor molecule on the target cell surface, and the method comprises: providing a sample, wherein the sample contains the target cell and a receptor-binding reagent, the receptor-binding reagent comprises a binding site B and a binding partner C, and the binding site B contained in the receptor-binding reagent can specifically bind to the receptor molecule; and A step of subjecting a sample to chromatography using a suitable stationary phase, wherein the stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix contains an affinity reagent, and the affinity reagent contains a binding site Z that specifically binds to binding partner C contained in a receptor binding reagent, whereby target cells are isolated. comprising.

[0016] According to a third aspect, the present invention provides a method for chromatographically isolating target cells from a sample, the target cells having receptor molecules on the surface of the target cells, the method comprising: providing a sample containing target cells; providing a receptor binding reagent comprising a binding site B and a binding partner C, wherein the binding site B contained in the receptor binding reagent can specifically bind to a receptor molecule on the surface of the target cell; wherein the binding partner C contained in the receptor binding reagent can reversibly bind to the binding site Z of the affinity reagent; and subjecting the sample to chromatography using a suitable stationary phase, wherein an affinity reagent is immobilized on the stationary phase; the affinity reagent contains a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor binding reagent, and the binding site B of the receptor binding reagent binds to a receptor molecule on the surface of the target cell, whereby the target cell is reversibly immobilized on the stationary phase; providing a competing reagent containing a binding site that specifically binds to the binding site Z of the affinity reagent; loading the competing reagent onto a first stationary phase, thereby disrupting the non-covalent reversible complex formed between the (multiple) receptor binding reagent, the receptor molecule, and the affinity reagent; recovering an eluted sample from the eluate of the first stationary phase, the eluted sample containing target cells; Subjecting the elution sample to chromatography on a suitable second stationary phase, wherein the second stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, and the gel filtration and / or affinity chromatography matrix comprises an affinity reagent having a binding site Z that specifically binds to binding partner C contained in the receptor-binding reagent, and Passing the elution sample through a second chromatography column comprising.

[0017] According to a fourth aspect, the present invention provides the use of a receptor-binding reagent and / or an affinity reagent for isolating target cells via chromatography using a stationary phase, wherein the target cells have receptor molecules on the surface of the target cells, the receptor-binding reagent comprises a binding site B and a binding partner C, the binding site of the receptor-binding reagent can specifically bind to the receptor molecules of the target cells, the dissociation constant (KD) for the binding of the receptor-binding reagent to the receptor molecules via the binding site B is of low affinity, or the dissociation rate constant (koff) for the binding of the receptor-binding reagent to the receptor molecules via the binding site B is about 3×10 -5 sec -1 or more, and the binding partner C contained in the receptor-binding reagent can reversibly bind to the binding site Z of the affinity reagent.

[0018] According to a fifth aspect, the present invention provides the use of one of streptavidin, streptavidin mutein (analog), avidin, and avidin analog for isolating target cells via chromatography, wherein the chromatography is gel filtration chromatography.

[0019] According to a sixth aspect, the present invention provides for the use of one of a chromatographic matrix of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, a polysaccharide grafted silica, a polyvinylpyrrolidone grafted silica, a polyethylene oxide grafted silica, a poly(2-hydroxyethyl aspartamide) silica, a poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of acrylate or acrylamide and diol, a copolymer of a polysaccharide and N,N'-methylenebisacrylamide, and any combination of two or more thereof for separating cells containing cell nuclei.

[0020] According to a seventh aspect, the present invention provides an arrangement of a first and a second stationary phase for chromatography, The first stationary phase is suitable for cell separation and is defined by an affinity chromatography matrix on which an affinity reagent is immobilized, the affinity reagent having at least one binding site Z capable of reversibly binding to a binding partner C included in a receptor binding reagent. The second stationary phase is suitable for separating target cells from other components and is a gel filtration matrix and / or an affinity chromatography matrix, the affinity chromatography matrix or the gel filtration and affinity chromatography matrix including an affinity reagent having a binding site Z that specifically binds to the binding partner C included in the receptor binding reagent. In some embodiments, the affinity reagents included in / immobilized on the first and second stationary phases are the same. In some embodiments, the affinity reagents included in / immobilized on the first and second stationary phases are streptavidin, streptavidin mutein, avidin, or avidin mutein.

[0021] According to an eighth aspect, the present invention provides a kit for isolating target cells, wherein the target cells have receptor molecules on the surface of the target cells, and the kit comprises (a) a receptor-binding reagent comprising a binding site B and a binding partner C, wherein the binding site B comprised in the receptor-binding reagent is capable of specifically binding to a receptor molecule on the surface of a target cell, and the binding partner C comprised in the receptor-binding reagent is capable of reversibly binding to a binding site Z on a multimerization reagent; and (b) a stationary phase suitable for cell separation, wherein the stationary phase is defined by a gel filtration matrix and / or an affinity chromatography matrix, and the affinity chromatography matrix or the gel filtration and affinity chromatography matrix comprises an affinity reagent having a binding site Z capable of reversibly binding to the binding partner C comprised in the receptor-binding reagent comprising the same.

[0022] According to a ninth aspect, the present invention provides a method for isolating target cells, wherein the target cells have receptor molecules on the surface of the target cells, and the method comprises providing a sample containing the target cells, providing a receptor-binding reagent comprising a monovalent binding site B and a binding partner C, wherein the receptor-binding reagent is selected from the group consisting of a monovalent antibody fragment, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule, wherein the monovalent binding site B comprised in the receptor-binding reagent is capable of specifically binding to a receptor molecule on the surface of a target cell, and the binding partner C comprised in the receptor-binding reagent is capable of reversibly binding to a binding site Z of an affinity reagent; and A step of subjecting a sample to chromatography on a suitable stationary phase, wherein an affinity reagent is immobilized on the stationary phase, the affinity reagent includes a binding site Z, the binding site Z reversibly binds to a binding partner C included in a receptor binding reagent, and a binding site B of the receptor binding reagent binds to a receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase comprising

[0023] According to a tenth aspect, the present invention provides an apparatus for purifying target cells, the apparatus comprising at least one arrangement of a first and a second stationary phase for chromatography. The first stationary phase of this arrangement is suitable for cell separation, the first stationary phase is an affinity chromatography matrix, and an affinity reagent is immobilized on the affinity chromatography matrix. The affinity reagent has at least one binding site Z capable of reversibly binding to a binding partner C included in a receptor binding reagent. The second stationary phase is suitable for cell separation, and the second stationary phase is a gel filtration matrix and / or an affinity chromatography matrix. The affinity chromatography matrix or the gel filtration and affinity chromatography matrix includes an affinity reagent having a binding site Z that specifically binds to the binding partner C included in the receptor binding reagent

[0024] According to an eleventh aspect, the present invention provides a method for screening target cells for the recombinant expression of a desired receptor molecule on the target cell surface, the desired receptor molecule being expressed on the target cell surface, the method comprising providing a sample, the sample comprising target cells that may be capable of recombinant expression of a desired target receptor providing a receptor binding reagent comprising a binding site B and a binding partner C The binding site B contained in the receptor binding reagent can specifically bind to a desired receptor molecule on the target cell surface, and the binding partner C contained in the receptor binding reagent can reversibly bind to the binding site Z of the affinity reagent. The process, and A step of subjecting the sample to chromatography on a suitable stationary phase, wherein an affinity reagent is immobilized on the stationary phase, the affinity reagent contains a binding site Z, and the binding site Z forms a reversible bond with the binding partner C contained in the receptor binding reagent. The binding site B of the receptor binding reagent binds to the receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase. Step including. [Invention 1001] A method for isolating a target cell, the target cell having a receptor molecule on the target cell surface, the method comprising: Providing a sample containing the target cell, Providing a receptor binding reagent comprising a binding site B and a binding partner C, The binding site B contained in the receptor binding reagent can specifically bind to a receptor molecule on the target cell surface, and the dissociation constant (K D ) is of low affinity, or the dissociation rate constant (koff) for the binding of the receptor binding reagent to the receptor molecule via the binding site B is about 3×10 -5 sec -1 or more, and the binding partner C contained in the receptor binding reagent can reversibly bind to the binding site Z of the affinity reagent. The process, and A step of subjecting the sample to chromatography on a suitable stationary phase, wherein an affinity reagent is immobilized on the stationary phase, The affinity reagent contains a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor binding reagent, and the binding site B of the receptor binding reagent binds to the receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase. Step A method comprising... [Invention 1002] The method of Invention 1001, wherein the affinity reagent comprises two or more binding sites Z that can reversibly bind to the binding partner C contained in the receptor-binding reagent. [Invention 1003] The method of Invention 1001, further comprising the step of loading a competitive reagent onto the stationary phase, wherein the competitive agent can disrupt the binding between the partner C of the receptor-binding reagent and the binding site Z of the affinity reagent, thereby substituting for the receptor-binding agent. [Invention 1004] The method of Invention 1003, wherein the competitive reagent can competitively bind to the binding site Z of the affinity reagent. [Invention 1005] Any of the methods of Invention 1001 - 1004, wherein the receptor-binding reagent is immobilized on the stationary phase before applying the sample containing the target cells to the stationary phase. [Invention 1006] Any of the methods of Invention 1001 - 1005, wherein the sample contains a mixture of target cells and additional cells, the additional cells lack the receptor molecule on their cell surface, and the method comprises the step of separating the target cells from the additional cells. [Invention 1007] The low-affinity interaction between the receptor-binding reagent and the receptor molecule has a K within the range of about 10 -3 ~ about 10 -7 M. D Any of the methods of Invention 1001 - 1006. [Invention 1008] A method for isolating target cells, wherein the target cells have a receptor molecule on the target cell surface, and the method comprises: providing a sample, the sample comprising target cells and a receptor-binding reagent, the receptor-binding reagent comprising a binding site B and a binding partner C, and the binding site B contained in the receptor-binding reagent can specifically bind to the receptor molecule, and A step of subjecting the sample to chromatography using a suitable stationary phase, wherein the stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, and the gel filtration and / or affinity chromatography matrix contains an affinity reagent, and the affinity reagent contains a binding site Z that specifically binds to binding partner C contained in the receptor binding reagent, whereby the target cells are isolated A method comprising [Invention 1009] The method according to any one of Inventions 1001 to 1008, wherein the chromatography is column chromatography or planar chromatography. [Invention 1010] The method according to Invention 1008, wherein the step of subjecting the sample to chromatography enables binding partner C contained in the receptor binding reagent to form a complex with binding site Z of the affinity reagent of the stationary phase to which it specifically binds, whereby the receptor binding reagent is immobilized on the stationary phase. [Invention 1011] The method according to Invention 1008 or 1010, wherein binding partner C contained in the receptor binding reagent can specifically bind to binding site Z of the affinity reagent, and the affinity reagent contains two or more binding sites Z that can specifically bind to binding partner C contained in the receptor binding reagent. [Invention 1012] The method according to Invention 1010 or 1011, wherein the affinity reagent is covalently immobilized on the affinity chromatography matrix and / or the gel filtration matrix. [Invention 1013] The method according to any one of Inventions 1008 to 1012, wherein the sample further contains a competing reagent, and the competing reagent binds to binding site Z of the affinity reagent that specifically binds to binding partner C contained in the receptor binding reagent, whereby the competing reagent is immobilized on the stationary phase. [Invention 1014] Further comprising a step of forming the sample, The step of forming the sample is providing a source sample comprising target cells, and adding a receptor-binding reagent to the source sample The method according to any one of 1001 to 1004 of the present invention. [The present invention 1015] The method according to 1014 of the present invention, wherein the source sample is expected to contain a plurality of target cells, and the source sample is contacted with a plurality of receptor-binding reagents, and an excess amount of the receptor-binding reagent is provided for the expected number of target cells. [The present invention 1016] The method according to any one of 1001 to 1015 of the present invention, wherein the sample is a fluid or contains a fluid. [The present invention 1017] The method according to any one of 1001 to 1016 of the present invention, wherein the sample contains a body fluid. [The present invention 1018] The method according to 1017 of the present invention, wherein the body fluid is blood or a blood component. [The present invention 1019] The method according to any one of 1001 to 1018 of the present invention, wherein the step of subjecting the sample to chromatography comprises passing the sample through a stationary phase of a chromatography column and washing the stationary phase with a fluid mobile phase, and the fluid mobile phase is essentially free of the receptor-binding reagent. [The present invention 1020] The method according to any one of 1001 to 1019 of the present invention, wherein the step of subjecting the sample to chromatography comprises eluting the target cells from a chromatography matrix. [The present invention 1021] The method according to 1020 of the present invention, further comprising the step of recovering the target cells. [The present invention 1022] A method for chromatographically isolating target cells from a sample, wherein the target cells have receptor molecules on the surface of the target cells, and the method comprises providing a sample comprising the target cells, providing a receptor-binding reagent comprising a binding site B and a binding partner C, Binding site B contained in the receptor binding reagent can specifically bind to receptor molecules on the target cell surface, The binding partner C contained in the receptor binding reagent can reversibly bind to the binding site Z of the affinity reagent, the step, and The step of subjecting the sample to chromatography on a suitable stationary phase, on which the affinity reagent is immobilized, The affinity reagent contains a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor binding reagent, and the binding site B of the receptor binding reagent binds to receptor molecules on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase, the step, The step of providing a competing reagent containing a binding site that specifically binds to the binding site Z of the affinity reagent; The step of loading the competing reagent onto a first stationary phase, Thereby, the non-covalent reversible complex formed between the (multiple) receptor binding reagent, the receptor molecule, and the affinity reagent is disrupted, the step; The step of recovering an eluted sample from the eluate of the first stationary phase, the eluted sample containing the target cell, the step; The step of subjecting the eluted sample to chromatography on a suitable second stationary phase, the second stationary phase being a gel filtration matrix and / or an affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix containing an affinity reagent having a binding site Z that specifically binds to the binding partner C contained in the receptor binding reagent, the step, and The step of passing the eluted sample through a second chromatography column A method comprising. [Inventive concept 1023] The method of Inventive concept 1022, wherein an affinity reagent is immobilized on an affinity chromatography matrix and / or a gel filtration matrix as the second stationary phase. [Inventive concept 1024] The method of the invention 1022 or 1023, wherein the step of passing the dissolution sample through the second stationary phase includes the step of immobilizing the competing reagent on the stationary phase of the second chromatography column by enabling the competing reagent to form a complex with the binding site Z of the affinity reagent. [The invention 1025] The method according to any one of inventions 1022 to 1024, wherein each of the first and second stationary phases is included in a column or is a planar stationary phase. [The invention 1026] The binding between the binding site B of the receptor-binding reagent and the receptor molecule has a dissociation constant (K -2 M) to about 10 -10 M within the range of D ), and the method according to any one of inventions 1022 to 1025. [The invention 1027] The dissociation constant (K D ) for the binding between the receptor-binding reagent and the receptor molecule via the binding site B is of low affinity, or the dissociation rate constant (koff) for the binding between the receptor-binding reagent and the receptor molecule via the binding site B is about 3×10 -5 sec -1 or more, and the method of the invention 1026. [The invention 1028] The reversible binding between the binding partner C of the receptor-binding reagent and the binding site Z of the affinity reagent has a dissociation constant (K -2 ) in the range of about 10 -13 to about 10 D M, and the method according to any one of inventions 1001 to 1027. [The invention 1029] The method according to any one of inventions 1001 to 1028, wherein the stationary phase is a non-magnetic material or a non-magnetizable material. [The invention 1030] The method of the present invention 1029, wherein the stationary phase comprises or consists of one of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, a polysaccharide grafted silica, a polyvinylpyrrolidone grafted silica, a polyethylene oxide grafted silica, a poly(2-hydroxyethyl aspartamide) silica, a poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of acrylate or acrylamide and diol, a copolymer of polysaccharide and N,N'-methylenebisacrylamide, and any combination of two or more thereof. [The present invention 1031] The method according to any one of the present inventions 1001 to 1030, wherein the affinity chromatography matrix and / or the gel filtration matrix comprises or consists of a monolithic matrix, a particulate matrix, or a planar matrix. [The present invention 1032] The method of the present invention 1031, wherein the particulate matrix has an average particle size of about 5 μm to about 200 μm or about 5 μm to 600 μm or about 5 μm to 1500 μm. [The present invention 1033] The method according to any one of the present inventions 1001 to 1032, wherein the affinity chromatography matrix and / or the gel filtration matrix has an average pore size of 0 to about 500 nm. [The present invention 1034] The method according to any one of the present inventions 1001 to 1033, wherein the receptor binding reagent is selected from the group consisting of an immunoglobulin, a functional fragment of an immunoglobulin, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule. [The present invention 1035] The method according to any one of the present inventions 1001 to 1034, wherein the binding partner C contained in the receptor binding reagent comprises one of biotin, a biotin analog, a streptavidin-binding peptide, and an avidin-binding peptide, and the affinity reagent comprises streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. [The present invention 1036] Any one of the methods of the present inventions 1001 to 1035, wherein the target cell is a mammalian cell. [The present invention 1037] Any one of the methods of the present inventions 1001 to 1036, wherein the target cell is a cell having a cell nucleus. [The present invention 1038] The method of the present invention 1037, wherein the target cell is a leukocyte or a stem cell. [The present invention 1039] The method of the present invention 1038, wherein the leukocyte is a lymphocyte. [The present invention 1040] Use of a receptor-binding reagent and / or an affinity reagent for isolating a target cell via chromatography using a stationary phase, wherein the target cell has receptor molecules on the surface of the target cell, the receptor-binding reagent comprises a binding site B and a binding partner C, the binding site of the receptor-binding reagent can specifically bind to the receptor molecules of the target cell, the dissociation constant (K D ) for the binding of the receptor-binding reagent to the receptor molecule via the binding site B is of low affinity, or the dissociation rate constant (koff) for the binding of the receptor-binding reagent to the receptor molecule via the binding site B has a value of about 3×10 -5 sec -1 or more, and the binding partner C contained in the receptor-binding reagent can reversibly bind to the binding site Z of the affinity reagent. [The present invention 1041] The use of the present invention 1040, wherein the affinity reagent comprises two or more binding sites Z that can reversibly bind to the binding partner C contained in the receptor-binding reagent. [The present invention 1042] The use of the present invention 1040 or 1041, wherein the receptor-binding reagent is one of an immunoglobulin, a functional fragment of an immunoglobulin, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule. [The present invention 1043] Use according to any one of the present inventions 1040 to 1042, wherein the binding partner contained in the receptor binding reagent includes one of biotin, biotin analog, streptavidin-binding peptide, and avidin-binding peptide, and the affinity reagent includes one of streptavidin, streptavidin analog, avidin, and avidin analog. [The present invention 1044] Use of streptavidin, streptavidin mtein, avidin, avidin mtein, or a mixture thereof for isolating target cells via chromatography, wherein the chromatography is gel filtration chromatography. [The present invention 1045] Use according to the present invention 1044, wherein the chromatography is column chromatography or planar chromatography. [The present invention 1046] Use according to the present invention 1044 or 1045, wherein the gel filtration chromatography is performed on a stationary phase on which streptavidin, streptavidin mtein, avidin, avidin mtein, or a mixture thereof is immobilized. [The present invention 1047] Use according to any one of the present inventions 1044 to 1046, wherein the target cell is a cell having a cell nucleus. [The present invention 1048] Use according to the present invention 1047, wherein the cell having a cell nucleus is a lymphocyte. [The present invention 1049] Use of a chromatographic matrix of one of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, a polysaccharide grafted silica, a polyvinylpyrrolidone grafted silica, a polyethylene oxide grafted silica, a poly(2-hydroxyethyl aspartamide) silica, a poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of acrylate or acrylamide and diol, a copolymer of polysaccharide and N,N'-methylenebisacrylamide, and any combination of two or more thereof for separating cells containing a cell nucleus. [The present invention 1050] Use of the present invention 1049, wherein the cell containing the cell nucleus is a lymphocyte. [The present invention 1051] Use of the present invention 1049 or 1050, wherein the copolymer of the polysaccharide and N,N'-methylenebisacrylamide is Sephacryl (registered trademark). [The present invention 1052] Use of the present invention 1049 or 1050, wherein the polysaccharide gel is Sepharose (registered trademark). [The present invention 1053] Use of the present invention 1049 or 1050, wherein the agarose gel is a crosslinked dextran gel such as Sephadex (registered trademark). [The present invention 1054] Use of the present invention 1049 or 1050, wherein the copolymer of acrylate and diol is Toyopearl (registered trademark). [The present invention 1055] Use of the present invention 1049 or 1050, wherein the polyacrylamide gel is one of Fractogel (registered trademark) and Bio-Gel (registered trademark). [The present invention 1056] Use of any one of the present inventions 1049 to 1055, wherein the chromatography gel is contained in a column. [The present invention 1057] Use of the present invention 1056, wherein the column is a cartridge. [The present invention 1058] Arrangement of the first and second stationary phases for chromatography, wherein the first stationary phase is suitable for cell separation, the first stationary phase is defined by an affinity chromatography matrix, and an affinity reagent is immobilized on the affinity chromatography matrix. The affinity reagent has at least one binding site Z that can reversibly bind to the binding partner C contained in the receptor binding reagent. The second stationary phase is suitable for cell separation, and the second stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, and the affinity chromatography matrix or the gel filtration and affinity chromatography matrix includes an affinity reagent having a binding site Z that specifically binds to binding partner C included in the receptor binding reagent, arrangement. [Invention 1059] The arrangement of Invention 1058, wherein at least the affinity reagent immobilized on the affinity chromatography matrix of the first stationary phase includes two or more binding sites Z capable of reversibly binding to binding partner C included in the receptor binding reagent. [Invention 1060] The arrangement of Invention 1058 or 1059, wherein each of the first and second stationary phases is included in a chromatography column or is a planar stationary phase. [Invention 1061] The arrangement according to any one of Inventions 1058 to 1060, including a plurality of first and second stationary phases. [Invention 1062] A kit of parts for isolating target cells, wherein the target cells have receptor molecules on the target cell surface, and the kit (a) A receptor binding reagent including binding site B and binding partner C, wherein the binding site B included in the receptor binding reagent can specifically bind to the receptor molecule on the target cell surface, and the binding partner C included in the receptor binding reagent can reversibly bind to the binding site Z on the multimerization reagent, receptor binding reagent; and (b) A stationary phase suitable for cell separation, wherein the stationary phase is defined by a gel filtration matrix and / or an affinity chromatography matrix, and the affinity chromatography matrix or the gel filtration and affinity chromatography matrix includes an affinity reagent having a binding site Z capable of reversibly binding to binding partner C included in the receptor binding reagent, stationary phase comprising, kit. [Invention 1063] A kit of parts of Invention 1062, wherein the stationary phase is included in a chromatography column or is a planar stationary phase. [Invention 1064] A kit of parts of Invention 1062 or 1063, comprising a second stationary phase suitable for cell separation / target cell separation from other components in a sample, the second stationary phase being defined by an affinity chromatography matrix on which an affinity reagent is immobilized, the affinity reagent comprising a binding site Z capable of specifically binding to binding partner C included in the receptor binding reagent. [Invention 1065] A kit of parts of Invention 1064, wherein the second stationary phase is included in a chromatography column or is a planar stationary phase. [Invention 1066] A method for isolating a target cell, the target cell having a receptor molecule on the target cell surface, the method comprising: providing a sample containing the target cell; providing a receptor binding reagent comprising a monovalent binding site B and a binding partner C, the receptor binding reagent being selected from the group consisting of a monovalent antibody fragment, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule; the monovalent binding site B included in the receptor binding reagent being capable of specifically binding to the receptor molecule on the target cell surface, and the binding partner C included in the receptor binding reagent being capable of reversibly binding to the binding site Z of the affinity reagent; and subjecting the sample to chromatography on a suitable stationary phase on which an affinity reagent is immobilized, the affinity reagent comprising a binding site Z that forms a reversible bond with the binding partner C included in the receptor binding reagent, and the binding site B of the receptor binding reagent binding to the receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase. A method comprising the above steps. [Invention 1067] The method of the present invention 1066, wherein the monovalent antibody fragment is a Fab fragment, an Fv fragment, or a single-chain Fv fragment. [The present invention 1068] The method of the present invention 1066, wherein the proteinaceous binding molecule having immunoglobulin-like function is selected from the group consisting of muteins based on polypeptides of the lipocalin family, globodies, proteins based on the ankyrin backbone, proteins based on the crystallin backbone, adnectins, and avimers. [The present invention 1069] Use of a receptor-binding reagent and / or an affinity reagent for isolating target cells via chromatography using a stationary phase, wherein the target cells have receptor molecules on the surface of the target cells, the receptor-binding reagent is selected from the group consisting of monovalent antibody fragments, proteinaceous binding molecules having immunoglobulin-like function, aptamers, and MHC molecules, the receptor-binding reagent comprises a binding site B and a binding partner C, and the binding site of the receptor-binding reagent can specifically bind to the receptor molecule of the target cells, Use, wherein the binding partner C comprised in the receptor-binding reagent can reversibly bind to the binding site Z of the affinity reagent. [The present invention 1070] An apparatus for purifying target cells, comprising at least one arrangement of the first and second stationary phases for chromatography as defined in the present invention 1058 - 1061. [The present invention 1071] The apparatus of the present invention 1070, further comprising a plurality of arrangements of the first and second stationary phases fluidly connected in series. [The present invention 1072] The apparatus of the present invention 1071, comprising a sample inlet fluidly connected to the first stationary phase of the first arrangement of the first and second stationary phases for chromatography. [The present invention 1073] An apparatus of the present invention 1072, comprising a sample outlet for purified target cells, the sample outlet being fluidly connected to a second stationary phase of a last arrangement of at least one of a first and a second stationary phase for chromatography. [The present invention 1074] An apparatus according to any one of the present inventions 1070 to 1073, comprising a competing reagent container fluidly connected to at least one of a first stationary phase of an arrangement of a first and a second stationary phase for chromatography. [The present invention 1075] A method for screening target cells for recombinant expression of a desired receptor molecule on the surface of the target cells, wherein the desired receptor molecule is expressed on the surface of the target cells, the method comprising: Providing a sample, the sample comprising target cells that may have the potential for recombinant expression of the desired target receptor; Providing a receptor binding reagent comprising a binding site B and a binding partner C, wherein the binding site B comprised in the receptor binding reagent is capable of specifically binding to the desired receptor molecule on the surface of the target cells, and the binding partner C comprised in the receptor binding reagent is capable of reversibly binding to a binding site Z of an affinity reagent; Subjecting the sample to chromatography on a suitable stationary phase, on which an affinity reagent is immobilized, wherein the affinity reagent comprises a binding site Z, the binding site Z forming a reversible bond with the binding partner C comprised in the receptor binding reagent, and the binding site B of the receptor binding reagent binds to a receptor molecule on the surface of the target cells, whereby the target cells are reversibly immobilized on the stationary phase; comprising. [The present invention 1076] The screening method of the present invention 1075, wherein the desired receptor molecule is endogenous or exogenous to the target cells. [The present invention 1077] The screening method of the present invention 1075 or 1076, wherein the target cells are transfected with a nucleic acid encoding the desired receptor molecule.

Brief Description of the Drawings

[0025] The present invention will be better understood with reference to the detailed description when considered in conjunction with non-limiting embodiments and the accompanying drawings. These drawings illustrate aspects of the method of the present invention. While not desiring to be bound by theory, these drawings include conclusions regarding the underlying separation mechanism. These conclusions are provided for illustrative purposes only and serve only to visualize how a surprising separation that can be achieved can be envisioned at the molecular level.

[0026]

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Mode for Carrying Out the Invention

[0027] Detailed Description of the Invention The present invention provides a method and apparatus for performing fluid chromatography separation of cells and other biological entities, such as organelles, viruses, liposomes, etc. (thus, references to target cells hereinafter include references to all other biological entities). A target cell or population of target cells is isolated, for example, from a sample that may contain various different cells or cell populations. Substantially any of said target cells having at least one common receptor molecule on its surface can be separated from other components contained in the sample. In order to achieve an avidity effect as discussed below for affinity chromatography as described herein, the receptor molecule is typically present in two or more copies on the surface of the target cell. As used herein, the term "(target) cell" encompasses all biological entities / vesicles in which a membrane (which may also include a lipid bilayer) separates its interior from its external environment (surrounding environment) and contains one or more specific receptor molecules on the surface of the biological entity / vesicle. This means that a target cell / biological entity / vesicle or population of target cells is defined by the presence of at least one common specific receptor molecule on its surface. As used herein, "isolated" means that the target cell is concentrated in the sample obtained as a result of the method of the present invention as compared to the content (concentration) of the sample for isolating the target cell. This means that the target cell can be concentrated in the sample from an approximate content of about 0.1% of the total cell amount in the sample to about 10% or more, 20% or more, 30% or more, 40% or more in the sample recovered by the method of the present invention. "Isolated" also means that the resulting sample contains the target cell as essentially the only type of cell (cell population), for example, the target cell is more than 75% or more than 80% or more than 85% or more than 90% or more than 95% or more than 97% or more than 99% of the cells present in the sample. "Isolated" also includes that the sample containing the target cell is free of reactants (e.g., receptor-binding reagents or competing reagents as defined herein) after the isolation / purification method of the present invention has been performed. The term "isolated" also includes the detection of the presence or absence of target cells in the sample.Thus, the isolation of target cells can be used for analytical or preparative purposes (e.g., not only for detecting the presence of a target cell population, but also for quantifying the cells present in a sample or for isolating large numbers of cells for cell-based therapy). Analytical purposes include diagnostic applications, as well as, for example, basic research applications in which the isolation method of the present invention is used for screening purposes to determine whether a particular receptor molecule, such as a G protein-coupled receptor (GPCR) or any other physiologically relevant receptor (e.g., the insulin receptor), is recombinantly expressed in a selected host cell (see also below).

[0028] In some embodiments, the cell can be a prokaryotic cell, such as a bacterial cell. The cell can be an archaeon in some embodiments. The cell can be a virus or an organelle, such as a mitochondrion, chloroplast, microsome, lysosome, Golgi apparatus, or nucleus in some embodiments. In some embodiments, the cell can be a eukaryotic cell, such as a plant cell, fungal cell, yeast cell, protozoan, or animal cell. The target cell includes a cell nucleus in some embodiments. In some embodiments, the target cell is a mammalian cell including cells of rodent species or cells of Lissamphibia, such as frog, Xenopus laevis, Japanese fire-bellied toad, or newt. Examples of mammalian cells include, but are not limited to, blood cells, sperm cells, or tissue cells, such as hepatocytes, or stem cells, such as CD34-positive peripheral stem cells derived from a suitable source or stem cells expressing Nanog or Oct-4. The blood cells can be, for example, white blood cells or red blood cells. The white blood cells can be, for example, neutrophils, eosinophils, basophils, monocytes, lymphocytes, macrophages, or dendritic cells. Each lymphocyte is, for example, merely illustrative, such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells (examples illustrative of memory T cells are specific central memory T cells of CD62L + CD8 + ), or regulatory T cells (examples illustrative of Tregs are CD4 +CD25 + CD45RA+ Treg cells), helper T cells, e.g., CD4 + It can be a T cell, B cell, or natural killer cell that includes helper T cells.

[0029] The fact that any other population of biological entities, a target cell population, or as described above, whose membrane (which can also be a lipid bilayer) separates its internal and external environments and further characterized by containing a common specific receptor molecule on its surface, can be purified by the method of the present invention in the subsequent removal of any purification reagent (receptor binding reagent; competitive reagent, affinity / multimerization reagent) used provides a regulatory advantage that the purification reagent is not administered to the patient while using such purified biological entities as a medicine, in addition to the advantage that the physiological state does not change when the target is a cell or an organelle. In such cases, the constraints required by regulatory authorities such as the FDA (USA) or EMEA (Europe) regarding the production process of the purification reagent are less costly than when the purification reagent is administered together with a medicine that is a cell or a liposome. Therefore, for example, when liposomes have to be purified and used as a medicine, there are obvious technical advantages to the method of the present invention for purifying entities whose physiological state should not be manipulated as in the case of such liposomes.

[0030] Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, monkeys, hamsters, gerbils, cats, ducks, American rabbits, armadillos, dogs, squirrel monkeys, goats, pigs, opossums, horses, elephants, bats, woodchucks, orangutans, rhesus monkeys, woolly monkeys, macaques, chimpanzees, tamarins (cotton-top tamarins (saguinus oedipus)), marmosets, and humans. The cells can be, for example, cells of a tissue, such as an organ or a part thereof. Examples of each organ include, but are not limited to, adrenal tissue, bone, blood, bladder, brain, cartilage, colon, eye, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, prostate, skin, small intestine, spleen, stomach, testis, thymus, tumor, vascular tissue or uterine tissue, or connective tissue. In some embodiments, the cells are stem cells.

[0031] The sample from which the target cells are isolated can be of any origin. The sample can be derived from, for example, but not limited to, humans, animals, plants, bacteria, fungi, or protozoa. Thus, but not limited to, soil samples, air samples, environmental samples, cell culture samples, bone marrow samples, rainfall samples, fallout samples, sewage samples, groundwater samples, erosion samples, archaeological samples, food samples, blood samples (including whole blood), serum samples, plasma samples, urine samples, fecal samples, semen samples, lymph fluid samples, cerebrospinal fluid samples, nasopharyngeal wash samples, sputum samples, oral swab samples, pharyngeal swab samples, nasal swab samples, bronchoalveolar lavage samples, bronchial secretion samples, milk samples, amniotic fluid samples, biopsy samples, cancer samples, tumor samples, tissue samples, cell samples, cell culture samples, cell lysate samples, virus culture samples, nail samples, hair samples, skin samples, forensic samples, infection samples, nosocomial infection samples, cosmic samples, or any combination thereof. If desired, each sample may be pretreated to any extent. By way of illustrative example, tissue samples may be digested, homogenized, or centrifuged prior to use in the methods according to the present invention. In another illustrative example, a sample of a body fluid such as blood can be obtained by standard isolation of blood cells. When the isolation methods described herein are used in basic research, the sample can be cells for in vitro cell culture experiments. The sample will typically be prepared in the form of a fluid, such as a solution or dispersion.

[0032] Generally, the chromatography method according to the present invention is liquid chromatography, typically liquid chromatography. This chromatography can be carried out in a flow-through mode in which a fluid sample containing the cells to be isolated is applied, for example by gravity flow or by a pump, to one end of a column containing a chromatography matrix, and the fluid sample exits the column from the other end of the column (see Examples 1-7 in this regard). Further, the chromatography can be carried out in an "up and down" mode in which a fluid sample containing the cells to be isolated is applied by a pipette to one end of a column containing a chromatography matrix filled inside, for example, a pipette tip, the fluid sample enters the chromatography matrix / pipette tip, and exits from the other end of the column (see Examples 8-10 in this regard). Alternatively, the chromatography can also be carried out in a batch mode in which the chromatography material (stationary phase) is incubated with the sample containing the cells, for example, while shaking, while rotating, or while repeatedly contacting, and then the fluid sample is taken out, for example, using a pipette. Any material can be used as the chromatography matrix in the context of the present invention as long as the material is suitable for the isolation of cells by chromatography. Suitable chromatography materials are at least essentially harmless, i.e., not harmful to cell viability (or viability or stability of biological entities) when used in a packed chromatography column under the desired conditions for cell isolation and / or cell separation. The chromatography matrix as used in the present invention remains in a predefined location, typically a predefined position, whereas the location of the sample to be separated and the components contained therein changes. Thus, in accordance with the standard understanding of those skilled in the art that the stationary phase is part of a chromatographic system in which the mobile phase flows (by flow-through or in batch mode) and the components contained in the liquid phase (dissolved or dispersed) are partitioned between the stationary phase and the liquid phase, the chromatography matrix is the "stationary phase".Accordingly, the terms "chromatography matrix" and "stationary phase" are used interchangeably herein. In this regard, it should be noted that particles such as freely mobile magnetic beads added to a liquid sample are mixed with the sample and then removed from the sample, for example, by discarding the supernatant (liquid) while temporarily holding those beads in place (e.g., by an external magnetic substance or centrifugation), but are not the stationary phase as used herein. Thus, a method in which such (magnetic) beads are added to a sample containing target cells for immobilization of the target cells on such beads (immobilization via a complex formed between the target cells and a receptor-binding reagent and an affinity / multimerization reagent), and then the beads are separated from the sample, for example, by temporarily holding those beads in place and discarding the supernatant, is not the method of the present invention.

[0033] Typically, each chromatographic matrix has a solid or semi-solid form, while the sample containing the target cells to be isolated / separated is in a fluid phase. Similarly, the mobile phase used to perform chromatographic separation is in a fluid phase. The chromatographic matrix can be a granular material (granular material of any suitable size and shape), or a monolithic chromatographic material including a paper substrate or membrane (see the Examples section). Thus, the present chromatography can be both column chromatography and planar chromatography. In addition to standard chromatographic columns, columns or pipette tips that allow two-way flow, such as PhyTip® columns available from PhyNexus, Inc., San Jose, CA, USA, can be used for column-based / flow-through mode-based chromatographic separation of cells as described herein. Thus, pipette tips or columns that allow two-way flow are also included in the term "chromatographic column" as used herein. When using a granular matrix material, the granular matrix material can have an average particle size of, for example, about 5 μm to about 200 μm or about 5 μm to about 400 μm or about 5 μm to about 600 μm. As will be described in detail below, the chromatographic matrix can be, or can include, for example, a polymer resin or a metal oxide or a semi-metal oxide. When planar chromatography is used, the matrix material can be any material suitable for planar chromatography, such as a conventional cellulose-based membrane or an organic polymer-based membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate. In one aspect, the chromatographic matrix / stationary phase is a non-magnetic material or a non-magnetizable material.

[0034] Non-magnetic or non-magnetizable chromatographic stationary phases that are used in the art and are also suitable in the present invention include derivatized silica or cross-linked gels. Cross-linked gels (typically produced in the form of beads) can be based on natural polymers, i.e., polymer classes that exist in nature. For example, the natural polymer on which the chromatographic stationary phase is based is a polysaccharide. Each polysaccharide is usually cross-linked. Examples of polysaccharide matrices are agarose gels (e.g., Superflow™ agarose or Sepharose® materials, e.g., Superflow™ Sepharose® available commercially in various bead sizes and pore sizes) or gels of cross-linked dextran. Further examples by way of illustration are granular cross-linked agarose matrices (of various bead sizes and having various pore sizes) with dextran covalently bound, available commercially as Sephadex® or Superdex®, both available from GE Healthcare. Another example by way of illustration of such chromatographic materials is Sephacryl®, available from GE Healthcare in various bead sizes and pore sizes.

[0035] Cross-linked gels can also be based on synthetic polymers, i.e., polymer classes that do not exist in nature. Usually, such synthetic polymers on which the chromatographic stationary phase for cell separation is based are polymers having polar monomer units, and thus the polymer is essentially polar. Such polar polymers are hydrophilic. Hydrophilic ("water-loving") molecules, also called lipophobic ("fat-hating"), contain moieties that can form dipole interactions with water molecules. Hydrophobic ("water-hating") molecules, also called lipophilic, tend to separate from water.

[0036] Examples illustrative of suitable synthetic polymers are polyacrylamide, styrene - divinylbenzene gels, and copolymers of acrylate and diol or acrylamide and diol. An illustrative example is a polymethacrylate gel commercially available as Fractogel®. A further example is a copolymer of ethylene glycol and methacrylate commercially available as Toyopearl®. In some embodiments, the chromatographic stationary phase can also include natural and synthetic polymer components, such as a composite matrix or composite or copolymer of a polysaccharide and agarose or a polysaccharide and N,N'-methylenebisacrylamide, such as a polyacrylamide / agarose composite. An illustrative example of a copolymer of dextran and N,N'-methylenebisacrylamide is the Sephacryl® series of materials described above. Derivatized silica can include silica particles bonded to a synthetic or natural polymer. Examples of such embodiments include, but are not limited to, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethyl aspartoamide) silica, and poly(N-isopropylacrylamide) grafted silica.

[0037] The chromatography matrix used in the present invention is, in some embodiments, for example, a gel filtration (also known as size exclusion) matrix when used in a removal cartridge as described herein. Gel filtration can be characterized by the property that it is designed to interact minimally, if at all, with the cells to be separated. Thus, the gel filtration matrix allows for the separation of cells or other biological entities generally based on their size as defined herein. Each chromatography matrix is typically a particulate porous material as described above. The chromatography matrix can have a specific exclusion limit, which is typically defined in terms of the molecular weight above which molecules are completely excluded from entering the pores due to their size. Each molecular weight defining the size exclusion limit can be selected to be less than the molecular weight corresponding to the molecular weight of the target cells (or biological entity) to be isolated. In such embodiments, the target cells are prevented from entering the pores of the size exclusion chromatography matrix. Similarly, the stationary phase, which is an affinity chromatography matrix, can have pores that are smaller in size than the size of the selected target cells. In an exemplary embodiment, the affinity chromatography matrix and / or the gel filtration matrix have an average pore size of from 0 to about 500 nm.

[0038] Other components present in the sample, such as receptor-binding molecules or competing reagents, may have a size smaller than the exclusion limit of the pores, which can enter the pores of the size-exclusion chromatography matrix. Among such components that can partially or completely enter the pore volume, larger molecules that are inaccessible due to the pore volume usually elute first, while the smallest molecules elute last. In some embodiments, the exclusion limit of the size-exclusion chromatography matrix is selected to be smaller than the maximum width of the target cells. Thus, components accessible to the pore volume will typically remain in or on the size-exclusion chromatography matrix for a longer time than the target cells. Accordingly, the target cells can be recovered separately into the eluate of the chromatography column from other substances / components of the sample. Thus, components such as receptor-binding reagents, or competing reagents where applicable, elute from the gel filtration matrix at a later time than the target cells. The separation effect will be further enhanced if the gel permeation matrix contains an affinity reagent (usually covalently bound on its matrix) that can bind to a binding site, such as binding site Z for reagents such as receptor-binding reagents and / or competing reagents present in the sample. The receptor-binding reagent and / or competing reagent binds to the binding site Z of the affinity reagent and is thereby immobilized on the gel permeation matrix. This method is typically carried out in a removal cartridge as used in the present invention, and in some embodiments, the methods, combinations, and kits according to the present invention include and / or use such a gel filtration matrix. In each method, the cells are appropriately separated based on size.

[0039] The chromatography matrix used in the present invention may also include a magnetically attractable substance, for example, one or more magnetically attractable particles or a magnetic fluid. Each magnetically attractable particle may include a multimerization reagent or an affinity reagent having a binding site capable of binding to a target cell. The magnetically attractable particles may include diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Superparamagnetic materials respond to a magnetic field by an induced magnetic field without generating a permanent magnetization. Magnetic particles based on iron oxide are commercially available, for example, as Dynabeads® from Dynal Biotech, as magnetic MicroBeads from Miltenyi Biotec, as magnetic porous glass beads from CPG Inc., and from various other sources, for example, to name a few, Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc. Magnetic nanoparticles based on superparamagnetic Co and FeCo and ferromagnetic Co nanocrystals have been reported, for example, by Hutten, A. et al. (J. Biotech. (2004), 112, 47-63). However, in some embodiments, the chromatography matrix used in the present invention does not have any magnetically attractable substances.

[0040] In some embodiments of the method of isolating target cells, the chromatography matrix is used as an affinity chromatography matrix. The affinity chromatography matrix itself contains a permanently bound (usually covalently bound) moiety that can specifically bind to a selected target. For example, a conventional affinity chromatography matrix may contain an antibody that binds to a particular given target. Alternatively, a chromatography matrix used for immobilized metal affinity chromatography (IMAC) is modified with a chelating ligand agent, such as trisiminodiacetic acid, or for example an oligohistidine tag, that can form a coordination bond between a metal ion and a particular exposed side chain of a protein. Thus, in the art, an affinity chromatography matrix is generally designed such that it can specifically bind to the analyte or target to be isolated. For example, in the "selection cartridge" as described in detail below, in the present invention using a chromatography matrix, the affinity chromatography matrix itself is not designed to specifically bind to the target cells to be isolated. Rather, in such embodiments, the affinity chromatography matrix (stationary phase) used in the present invention comprises an affinity reagent having at least one or more binding sites Z that can specifically bind to the receptor binding reagent also used in the present invention. When the receptor binding reagent is contacted with the affinity / multimerization reagent, a reversible complex is formed via the binding partner C of the receptor binding reagent and one or more binding sites Z of the affinity / multimerization reagent. Thus, the formation of this complex depends on the non-covalent interaction between the ligand and its respective binding partner, and is thus essentially different from the use of cleavable covalent bonds as described by Bonnafous et al. supra.As long as the affinity reagent is present / provided on the affinity chromatography matrix at a surface density high enough to produce an avidity effect when a complex of a receptor-binding reagent and an affinity reagent is formed via a binding site and a binding partner C, it is usually sufficient for the affinity reagent to contain one binding site Z that can form a reversible bond with the binding partner C. However, it is also possible for the affinity reagent to contain two or more binding sites Z for the binding partner C. In the non-covalently bound complex formed, when a target cell having (at least two copies of) receptor molecules is present in the sample and is contacted with a receptor-binding reagent having one or more binding sites B that can bind to a specific receptor molecule, two or more receptor-binding reagents are immobilized on the affinity chromatography matrix in a state where they are arranged close to each other so that an avidity effect can occur. Thus, in these embodiments, an avidity (multimerization) effect similar to that described in U.S. Patent No. 7,776,562, U.S. Patent No. 8,298,782, or International Patent Application WO02 / 054065 can occur to enable reversible immobilization of the target cell on the affinity chromatography matrix. The binding between the binding site Z of the affinity reagent (which can also act as a multimerizing agent thereafter) and the binding partner C of the receptor-binding reagent can be disrupted by the addition of a competing agent, so that the target cell can then be eluted under mild conditions where the receptor-binding reagent dissociates completely from the target cell, thereby avoiding the receptor-binding reagent affecting the functional state of the target cell. Thus, this isolation of the target cell via this affinity chromatography method not only has the advantage of enabling the isolation / purification of a target cell population (or any other biological entity described herein) without changing the functional state of the target cell population defined by a common specific receptor molecule.Rather, this method completely eliminates the need to use magnetic beads for cell purification, thereby simplifying any further cell manipulation and having the additional advantage of opening the way to the automation of the isolation of target cells as described herein.

[0041] In another aspect of the method according to the present invention, a chromatography matrix to which an affinity reagent is immobilized is used. The affinity reagent can bind to binding partner C included in the receptor binding reagent (see below). Such a chromatography matrix can be an affinity chromatography matrix. It can also be a gel filtration matrix to which an affinity reagent is bound. In some aspects, the chromatography matrix is included in a chromatography column, for example, packed in the column. By using the immobilized affinity reagent, the chromatography matrix can deplete the mobile phase of the receptor binding reagent. Similarly, the receptor binding reagent can be depleted from a sample that has contacted the chromatography matrix, for example, a sample loaded onto a column packed with the matrix. In one method according to the present invention, the receptor binding reagent is included in each stationary phase, i.e., the sample that is contacted with the chromatography matrix.

[0042] After applying a sample containing the target cells, the chromatography matrix (regardless of whether it is used for affinity chromatography or for gel permeation) can subsequently be washed with a mobile phase, such as an aqueous medium, such as a buffer, to remove any substances not immobilized on the chromatography matrix. The formation of the non-covalent complex described above immobilizes the target cells on the affinity chromatography matrix, and the dissociation of the complex can be induced, for example, by changing the conditions. Such a change in conditions can be, for example, a change in the ionic strength of the aqueous mobile phase or a change in temperature. In some embodiments, a competing reagent is used to induce the dissociation of the reversible non-covalent complex between the receptor, the receptor-binding reagent, and the affinity reagent. The competing reagent can associate with the affinity reagent by occupying or blocking the binding site of the affinity reagent for the binding partner contained in the receptor-binding reagent. By using a competing reagent that has a particularly high affinity for the affinity reagent, or by using an excess amount of the competing reagent with respect to at least one of the target cells and the receptor-binding reagent (in which case the competing reagent may have a lower affinity for the binding site Z of the affinity reagent than for the binding partner C of the receptor-binding reagent), the non-covalent bond between the receptor-binding reagent and the multimerization reagent can be disrupted. The target cells are eluted from a chromatography matrix, such as a column filled with the chromatography matrix. The eluate is collected, whereby the target cells are recovered.

[0043] In some embodiments, a source sample suspected of containing or containing target cells is used, to which a receptor binding reagent has been added to form the non-covalent complex described above containing the target cells and the affinity reagent on the affinity chromatography matrix. By way of illustrative example, a blood sample (e.g., a whole blood sample) or a lymph sample may define such a source sample (see the Examples section). A receptor binding reagent having a binding site for the desired target cells present in the blood or lymph may be selected respectively. That receptor binding reagent, optionally, a buffer may also be added to the blood sample or lymph sample. The buffer used is used to equilibrate the chromatography matrix and may be at least essentially the same as the buffer used for subsequent washing. Subsequently, the sample may be loaded onto a chromatography column. In this chromatography column, an affinity reagent capable of binding to the receptor binding reagent may be immobilized on the matrix. Alternatively, the receptor binding reagent may already be immobilized on the affinity chromatography matrix before applying the sample of target cells to the affinity chromatography matrix. After the sample, e.g., a blood or lymph sample, is completely loaded onto the chromatography column, optionally together with the receptor binding reagent, the chromatography matrix may be washed with the mobile phase. Then, a competing reagent that may be included in the buffer used for washing the chromatography matrix may be loaded onto the chromatography column. Subsequently, the chromatography matrix may be washed with the mobile phase. Elution of the target cells may be monitored using standard detection techniques such as an optical detection device. Then, the target cells may be recovered. Thus, such an eluate may contain the receptor binding reagent and / or the competing reagent.

[0044] Since such eluates of target cells can be further purified, the chromatography matrix (e.g., a size exclusion chromatography matrix) can include a molecule immobilized on the chromatography matrix that specifically binds to a binding partner B included in an affinity reagent, e.g., a receptor-binding reagent, and / or a binding site Z that can specifically bind to a competing reagent.

[0045] Thus, in accordance with the above, an affinity reagent may be immobilized on the size exclusion chromatography matrix used herein. Since each chromatography matrix can also separate substances according to size and / or shape, it can be treated as a mixed-mode chromatography matrix. Thus, in an aspect where the affinity reagent does not match the receptor-binding reagent, the mixed-mode chromatography matrix can still be used as a size exclusion chromatography matrix in that the affinity reagent immobilized on such a size exclusion chromatography matrix has a binding site that cannot form a complex with the selected receptor-binding reagent. In an aspect where the immobilized affinity reagent has a binding site capable of forming a complex with the selected receptor-binding reagent, the affinity reagent can serve to reversibly immobilize target cells on the chromatography matrix.

[0046] The fluid phase used as the mobile phase in chromatography can be any fluid suitable for preserving the biological activity of the target cells. Typically, the fluid is a liquid. In some embodiments, each liquid is in the form of or contains water, for example, in the form of an aqueous solution. Additional components may be included in each aqueous solution, for example, dissolved or suspended therein. By way of illustrative example, the aqueous solution may contain one or more buffer compounds. Numerous buffer compounds are used in the art and can be used to perform the various processes described herein. Examples of buffers include solutions of phosphates, such as phosphate buffered saline (PBS), carbonates, succinates, carbonates, citrates, acetates, formates, barbiturates, oxalates, lactates, phthalates, maleates, cacodylates, borates, N-(2-acetamido)-2-amino-ethanesulfonate (also called ACES), N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (also called HEPES), 4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (also called HEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also called PIPES), (2-[tris(hydroxymethyl)-methylamino]-1-ethanesulfonic acid (also called TES), 2-cyclohexylamino-ethanesulfonic acid (also called CHES), and solutions of N-(2-acetamido)-iminodiacetate (also called ADA), but are not limited thereto. Any counterion can be used in these salts; ammonium, sodium, and potassium can serve as illustrative examples. Further examples of buffers include, by way of a few examples, tri-ethanolamine, diethanolamine, zwitterionic buffers, such as betaine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris-(hydroxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane (also called BIS-TRIS), and N-[tris(hydroxymethyl)-methyl]-glycine (also called TRICINE), but are not limited thereto.The buffer may further contain components that stabilize the target cells to be isolated, such as proteins, such as (serum) albumin, growth factors, trace elements, and the like. The selection of a suitable mobile phase is within the knowledge of those skilled in the art and can be done empirically.

[0047] In accordance with the co-pending international patent application PCT / EP2012 / 063969, published as WO2013 / 011011, the entire content of which is incorporated herein by reference for all purposes, the strength of the binding between the receptor binding reagent and the receptor molecule on the target cell may not be essential for the reversibility of the binding of the target cell to the affinity reagent via the receptor binding reagent. Rather, the dissociation constant (K d ) for the binding between the receptor binding reagent and the receptor molecule via binding site B, regardless of the strength of the binding, means that it is within the range of low affinity, for example, about 10 -3 to about 10 -7 M of K d or within the range of high affinity, for example, about 10 -7 to about 1×10 -10 M of K d , as long as the dissociation of the binding between the receptor binding reagent and the receptor molecule via binding site B occurs fast enough, the target cells can be reversibly stained. In this regard, the dissociation rate constant (k off ) for the binding between the receptor binding reagent and the receptor molecule via binding site B can have a value of about 3×10 -5 sec -1 or higher (this dissociation rate constant is a constant that characterizes the dissociation reaction of the complex formed between the binding site B of the receptor binding reagent and the receptor molecule on the surface of the target cell). The association rate constant (k on ) for the association reaction between the binding site B of the receptor binding reagent and the receptor molecule on the surface of the target cell can have any value. To ensure a sufficiently reversible binding between the receptor molecule and the receptor binding reagent, about 3×10 -5 sec -1 or higher, about 5×10 -5 sec -1 or higher, for example, about 1×10 -4 sec -1Above, about 1.5×10 -4 sec -1 Above, about 2.0×10 -4 sec -1 Above, about 2.5×10 -4 sec -1 Above, about 3×10 -4 sec -1 Above, about 3.5×10 -4 sec -1 Above, about 4×10 -4 sec -1 Above, about 5×10 -4 sec -1 Above, about 7.5×10 -4 sec -1 Above, about 1×10 -3 sec -1 Above, about 1.5×10 -3 sec -1 Above, about 2×10 -3 sec -1 Above, about 2.5×10 -3 sec -1 Above, about 3×10 -3 sec -1 Above, about 4×10 -3 sec -1 , about 5×10 -3 sec -1 Above, about 7.5×10 -3 sec -1 Above, about 1×10 -2 sec -1 Above, about 5×10 -2 sec -1 Above, about 1×10 -1 sec -1 Above, or about 5×10 -1 sec -1 It is beneficial to select the k value of the binding equilibrium having the above value. The term "about" refers to the k off rate, the k off rate, or the K on rate, or the K D(See below) When used in this specification with respect to, it is meant to include a tolerance of ±20.0% including ±15.0%, ±10.0%, ±8.0%, ±9.0%, ±7.0%, ±6.0%, ±5.0%, ±4.5%, ±4.0%, ±3.5%, ±3.0%, ±2.8%, ±2.6%, ±2.4%, ±2.2%, ±2.0%, ±1.8%, ±1.6%, ±1.4%, ±1.2%, ±1.0%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, or ±0.01%. Here, note that the values of the kinetic and thermodynamic constants used in this specification refer to the conditions of atmospheric pressure, i.e., 1.013 bar and room temperature, i.e., 25°C.

[0048] When the receptor-binding reagent is represented by the symbol "A", the receptor on the surface of the target cell is represented by the symbol "B", and the complex of the receptor-binding reagent and the receptor is represented by the symbol "AB", the bimolecular interaction between the receptor-binding reagent and the receptor is TIFF0007710427000001.tif18128 can be described by a two-state process. The dissociation K d constant of this process is TIFF0007710427000002.tif15128 as defined. In these equations, [A], [B], and [AB] are the equilibrium molar concentrations of the receptor, the receptor-binding reagent (ligand), and their respective complexes at a given temperature and a given pressure. The dissociation K d constant is also the constant of the association rate (k on ) for the rate of association / formation of the complex, also called the association rate constant, and the constant of the dissociation rate (k off ) for the dissociation of the complex, also called the dissociation rate constant, as TIFF0007710427000003.tif5128. In this regard, the dissociation constant K dNote that it defines a state of reaching equilibrium. However, equilibrium may not occur under the conditions of chromatographic separation. In some aspects, the reversible binding can be 3×10 -5 sec -1 or more in the context of the present invention, that is, it can be determined that it can be at least 3×10 -1 sec -5 sec -1 equal to. The reason can be explained by the dissociation rate constant (k d ) rather than the dissociation constant K off .

[0049] In some aspects, the receptor binding reagent has a single (monovalent) binding site B that can specifically bind to the receptor molecule. In some aspects, the receptor binding reagent has at least two binding sites B (i.e., a plurality of binding sites B including 3, 4, or 5 identical binding sites B) that can bind to the receptor molecule. In any of these aspects, the binding of the receptor molecule through each of the binding sites B can have a k -5 sec -1 value of 3×10 off or more. Therefore, the receptor binding reagent can be monovalent (e.g., a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or otherwise), e.g., a mutein based on a polypeptide of the lipocalin family (also known as Anticalin®), or a divalent molecule, e.g., an antibody, or a fragment in which both binding sites are retained, e.g., an F(ab')2 fragment. In some aspects, if the k off rate is 3×10 -5 sec -1 or more, the receptor molecule can be a multivalent molecule, e.g., a pentameric IgE molecule.

[0050] In some aspects of the present invention, providing for the (trace-free) isolation of biological materials via the reversible cell affinity chromatography techniques described herein is, at the molecular level, not at least the binding of a receptor-binding reagent via binding site B to a receptor molecule on a target cell at a rate of (3×10 -5 sec -1 or greater) k off rate. Rather, and as described, for example, in U.S. Patent No. 7,776,562 or International Patent Application WO02 / 054065, low-affinity binding of a receptor molecule to the binding site B of a binding receptor-binding reagent, with an avidity effect mediated by an immobilized affinity reagent, enables the reversible isolation of target cells without leaving a trace. In these aspects, a complex of two or more binding sites Z of the affinity reagent and the binding partner C of at least two receptor-binding reagents can be formed, thereby enabling reversible immobilization and subsequent elution of the target cells from the affinity chromatography matrix (via the addition of a competing agent that breaks the bond (complex) formed between the binding partner C and the binding site Z and dissociates the receptor-binding reagent from the target cell). As described above, such low binding affinity can be characterized by a dissociation constant (K D ) in the range of about 1.0×10 -3 M to about 1.0×10 -7 M.

[0051] The method according to the invention can be used, in some embodiments, to deplete a sample of reagents previously used in cell separation. Receptor binding reagents and competing agents can be present, for example, in the eluate of an affinity chromatography method in the form of a selection cartridge as described above. When using the method according to the invention, such reagents can be removed, at least essentially, from a sample, for example a cell population, which includes complete removal. As an illustrative example, a receptor binding reagent as defined above can be depleted from a sample to levels below the detection limit of, for example, FACS or Western blot. A competing reagent can potentially be used to elute target cells from an affinity purification medium such as affinity chromatography beads. This competing reagent has a binding site that can specifically bind to the binding site Z of the affinity reagent. In such embodiments, each method of the invention can be useful in their depletion, including the removal of receptor binding reagents and competing reagents.

[0052] In some embodiments, a method of isolating target cells can include two purification steps, and only the second step of those steps, i.e., the removal of receptor binding reagents and / or competing reagents in the "removal cartridge", is performed in accordance with the present invention. The first step can be a method of isolating target cells as described in U.S. Patent No. 7,776,562, U.S. Patent No. 8,298,782, or International Patent Application WO02 / 054065. Then, by performing the "removal method" according to the present invention on such a sample, additional cells as well as receptor binding reagents and competing reagents can be depleted from the target cell sample. Similarly, a sample obtained in the first step according to U.S. Patent No. 7,776,562, U.S. Patent No. 8,298,782, or International Patent Application WO02 / 054065 can also be subjected to gel permeation chromatography as described above using an unmodified chromatography matrix to which the affinity reagent is not immobilized. The first isolation step can also be any other known conventional technique method for cell isolation, for example, the method described in Example 11 of U.S. Patent No. 6,022,951, and then the isolated cells are subjected to a purification method as performed in the removal cartridge of the present invention.

[0053] Receptor molecules located on the surface of the target cells (or the accessible surface of the biological entity) can be any molecule as long as they remain covalently or non-covalently bound to the cell surface during the chromatography separation process in the method according to the present invention. The receptor molecule is a molecule to which a receptor-binding reagent can be directed. In some embodiments, the receptor is a peptide or protein, such as a membrane receptor protein. In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. The receptor that is a protein can be a surface membrane protein or an integral membrane protein. The protein can, in some embodiments, have one or more domains spanning the membrane. As some illustrative examples, membrane proteins having transmembrane domains include G-protein coupled receptors, such as odorant molecule receptors, rhodopsin receptors, rhodopsin pheromone receptors, peptide hormone receptors, taste receptors, GABA receptors, opioid receptors, serotonin receptors, Ca 2+ receptors, melanopsin, neurotransmitter receptors, such as acetylcholine, nicotine, adrenaline, noradrenaline, catecholamine, L-DOPA-, dopamine, and serotonin (biogenic amines, endorphin / enkephalin) neuropeptide receptors, ligand-gated, voltage-gated, or mechanically gated receptors, receptor kinases, such as serine / threonine kinases, tyrosine kinases, porins / channels, such as chloride ion channels, potassium channels, sodium channels, OMP proteins, ABC transporters (ATP-binding cassette transporters), such as amino acid transporters, Na-glucose transporters, Na + / iodide transporters, ion transporters, such as light-harvesting complexes, cytochrome c oxidase, ATPases Na / K, H / K, Ca, cell adhesion receptors, such as metalloproteases, integrins, or catherin.

[0054] In some embodiments, the receptor molecule is an antigen that defines a desired cell population or subpopulation, such as blood cells, such as lymphocytes (e.g., T cells, helper T cells, such as CD4 + helper T cells, B cells, or natural killer cells), monocytes, or stem cells, such as a population or subpopulation of CD34-positive peripheral stem cells or stem cells expressing Nanog or Oct-4. Examples of T cells include CMV-specific CD8 + T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). Examples illustrative of Tregs are CD4 + CD25 + CD45RA Treg cells, and an example illustrative of memory T cells is a specific central memory T cell of CD62L + CD8+. The receptor can also be a marker for tumor cells.

[0055] As shown above, the receptor-binding reagent has a binding partner C in addition to the binding site B that can bind to the receptor molecule. This binding partner C can bind to the binding site Z of the affinity reagent, and the multimerization reagent has one or more binding sites for the binding partner C. The non-covalent bond formed between the binding partner C contained in the receptor-binding reagent and the binding site Z of the affinity reagent can have any desired strength and affinity as long as it is breakable or reversible under the conditions under which the method of the present invention is performed. The dissociation constant (K D ) between the binding partner C contained in the receptor-binding reagent and the binding site Z of the affinity reagent can have a value in the range of about 10 -2 M to about 10 -13 M. Thus, this reversible bond is, for example, about 10 -2 M to about 10 -13 M or about 10 -3 M to about 10 -12 M or about 10 -4 M to about 10 -11 M or about 10 -5 M to about 10 -10 M of K Dmay have. The K of this binding D , as well as the K of the binding formed between binding site B of the receptor-binding reagent and the receptor molecule D , k off , and k on rates can be measured by any suitable means, such as fluorescence titration, equilibrium dialysis, or surface plasmon resonance. The receptor molecule-binding reagent may contain at least one second binding partner C, including two, three, or more, and the affinity reagent may contain at least two, such as 3, 4, 5, 6, 7, 8 or more binding sites for the binding partner contained in the receptor molecule-binding reagent. As described in U.S. Patent No. 7,776,562, U.S. Patent No. 8,298,782, or International Patent Application WO2002 / 054065, as long as binding partner C and binding site Z of the affinity agent can reversibly bind or multimerize in a (multivalent) complex, thereby causing an avidity effect, any combination of binding partner C and an affinity agent having one or more corresponding binding sites Z can be selected.

[0056] The binding partner contained in the receptor-binding reagent can be, for example, hydrocarbon-based (including polymers), and can contain nitrogen, phosphorus, sulfur, carbon, halogen, or pseudohalogen groups. The binding partner can be an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, sugar, oligosaccharide, or polysaccharide. As a further example, the binding partner can also be a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. Generally, such a binding partner has a higher affinity for the binding site of the multimerization reagent than other substances. Examples of each binding partner include, but are not limited to, crown ethers, immunoglobulins, fragments thereof, and proteinaceous binding molecules having antibody-like functions.

[0057] In some embodiments, the binding partner C included in the receptor binding reagent comprises biotin, and the affinity reagent comprises a streptavidin analog or an avidin analog that binds reversibly to biotin. In some embodiments, the binding partner C included in the receptor binding reagent comprises a biotin analog that binds reversibly to streptavidin or avidin, and the affinity reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that binds reversibly to each biotin analog. In some embodiments, the binding partner C included in the receptor binding reagent comprises a streptavidin or avidin binding peptide, and the affinity reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that binds reversibly to each streptavidin or avidin binding peptide.

[0058] In some embodiments, the binding partner included in the receptor binding reagent can comprise the streptavidin binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, and the affinity reagent can comprise streptavidin mutein (analog) Val44-Thr45-Ala46-Arg47 or streptavidin mutein (analog) Ile44-Gly45-Ala46-Arg47, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available as the trademark Strep-Tactin®. The streptavidin binding peptide can be, for example, a single peptide, such as the "Strep-tag®" described in U.S. Patent No. 5,506,121, or a streptavidin binding peptide having a sequential arrangement of two or more individual binding modules as described in International Patent Publication WO02 / 077018 or U.S. Patent No. 7,981,632.

[0059] In some embodiments, the binding partner C of the receptor-binding reagent comprises a moiety known to those skilled in the art as an affinity tag. In such embodiments, the affinity reagent comprises a corresponding binding partner known to bind to the affinity tag, such as an antibody or antibody fragment. Some examples illustrative of known affinity tags include, as a binding partner contained in the receptor-binding reagent, 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, HA-tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala), VSV-G-tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys), HSV-tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp), T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose-binding protein (MBP), the HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of the herpes simplex virus glycoprotein D, the "myc" epitope of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu of the transcription factor c-myc, V5-tag (sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr), or glutathione-S-transferase (GST). In such embodiments, the complex formed between one or more binding sites of the affinity reagent, in this case an antibody or antibody fragment and an antigen, can be competitively disrupted by adding free antigen, i.e., free peptide (epitope tag) or free protein (e.g., MBP or CBP). The affinity tag can also be an oligonucleotide tag.Such oligonucleotide tags can be used to hybridize, for example, to an oligonucleotide having a complementary sequence linked to or included in an affinity reagent.

[0060] Further examples of suitable binding partners include, but are not limited to, lectins, Protein A, Protein G, metals, metal ions, nitrilotriacetic acid derivatives (NTA), RGD-motifs, dextran, polyethyleneimine (PEI), redox polymers, glycoproteins, aptamers, dyes, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligo-dT. Lectins such as Concavalin A are known to bind polysaccharides and glycosylated proteins. Illustrative examples of dyes are triazine dyes such as Cibacron blue F3G-A (CB) or Red HE-3B that specifically bind to NADH-dependent enzymes. Green A binds to CoA protein, human serum albumin, and dehydrogenase. The dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. Metal cations such as Ni, Cd, Zn, Co, or Cu are typically used to bind to affinity tags such as oligohistidine-containing sequences including hexahistidine or His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tags (MAT tags), and N-methacryloyl-(L)-cysteine methyl ester.

[0061] In some embodiments, the binding between the binding partner C contained in the receptor binding reagent and one or more binding sites of the affinity reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the affinity / multimerization reagent typically comprises a divalent, trivalent, or tetravalent cation that is retained, e.g., complexed, by a suitable chelating agent. The binding partner contained in the receptor binding reagent may, in such embodiments, comprise, for example, a moiety containing a complex, divalent, trivalent, or tetravalent cation. Examples of each metal chelating agent include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), porphyrin, and heme. By way of example, EDTA forms complexes with most monovalent, divalent, trivalent, and tetravalent metal ions, such as silver (Ag + ), calcium (Ca 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co 3+ ), and zirconium (Zr 4+ ), while BAPTA is specific for Ca 2+ . By way of illustrative example, a standard method used in the art is the formation of a complex between an oligo-histidine tag and copper (Cu 2+ ), nickel (Ni 2+ ), cobalt (Co 2+ ), or zinc (Zn 2+ ) ions provided by the chelating agent nitrilotriacetic acid (NTA).

[0062] In some embodiments, binding partner C included in the receptor binding reagent comprises a calmodulin binding peptide, and the affinity reagent comprises, for example, multimeric calmodulin as described in U.S. Patent No. 5,985,658. In some embodiments, binding partner C included in the receptor binding reagent comprises a FLAG peptide, and the affinity reagent comprises an antibody that binds to the FLAG peptide, for example, the FLAG peptide that binds to monoclonal antibody 4E11 as described in U.S. Patent No. 4,851,341. In one embodiment, binding partner C included in the receptor binding reagent comprises an oligo-histidine tag, and the affinity reagent comprises an antibody or a transition metal ion that binds to the oligo-histidine tag. Disruption of all of these bound complexes can be achieved by chelation of metal ions, for example, calcium chelation, for example, by addition of EDTA or EGTA (supra). Antibodies such as calmodulin, 4E11, or chelated metal ions or free chelating agents can be multimerized by conventional methods in the first step, for example, biotinylation and complex formation with streptavidin or avidin or their multimers, or essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137, introduction of carboxyl residues into a polysaccharide, for example, dextran, and in the second step, by linking calmodulin or an antibody or a chelated metal ion or a free chelating agent via a primary amino group to a carboxyl group in a polysaccharide, for example, the dextran backbone, using conventional carbodiimide chemistry. In such embodiments, the bond between binding partner C included in the receptor binding reagent and one or more binding sites Z of the multimerization reagent can be disrupted by chelation of metal ions. Such metal chelation can be achieved, for example, by addition of EGTA or EDTA.

[0063] In some embodiments, the affinity reagent is an oligomer or polymer of streptavidin or avidin or any analog of streptavidin or avidin. The binding site Z is natural biotin that binds to avidin or streptavidin. Each oligomer or polymer can be cross-linked by a polysaccharide. In one embodiment, the oligomer or polymer of streptavidin or avidin or an analog of streptavidin or avidin is prepared by introduction of carboxyl residues into a polysaccharide, such as dextran, essentially as described in Noguchi, A, et al, Bioconjugate Chemistry (1992) 3, 132-137, in a first step. Then, streptavidin or avidin or an analog thereof can be linked in a second step via internal lysine residues and / or free N-terminal primary amino groups to carboxyl groups in the dextran backbone using conventional carbodiimide chemistry. Nevertheless, the cross-linked oligomer or polymer of streptavidin or avidin or any analog of streptavidin or avidin can also be obtained by cross-linking via a bifunctional molecule that acts as a linker, such as glutaraldehyde, or by other methods reported in the art.

[0064] In the method of the present invention, one or more binding sites of a receptor molecule-binding reagent that specifically binds to a receptor molecule can be, for example, an antibody, a fragment thereof, and a proteinaceous binding molecule having antibody-like functions. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2'-fragments, diabodies, triabodies (Iliades, P., et al, FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al, Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, L.J., et al, Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, one or more binding sites of the receptor molecule-binding reagent can be a bivalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein also known as "duocalin". In some embodiments, the receptor-binding reagent can have a single second binding site, i.e., can be monovalent. Examples of monovalent receptor-binding reagents include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules having antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv) including bivalent single-chain Fv fragments.

[0065] As described above, examples of proteinaceous binding molecules having antibody-like functions are muteins based on polypeptides of the lipocalin family (see, for example, WO03 / 029462, Beste et al, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903). Lipocalins, such as bilin-binding protein, human neutrophil gelatinase-associated lipocalin, human apolipoprotein D, or human tear lipocalin, have natural ligand-binding sites that can be modified to bind to a given target. Further examples of proteinaceous binding molecules having antibody-like binding properties that can be used as receptor-binding reagents that specifically bind to receptor molecules include so-called glubodies (see, for example, international patent application WO96 / 23879), ankyrin scaffolds (Mosavi, L.K., et al, Protein Science (2004) 13, 6, 1435-1448) or proteins based on crystalline scaffolds (for example, international patent application WO01 / 04144), proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, AdNectins, tetranectins, and avimers, but are not limited thereto. Avimers, including multivalent avimer proteins evolved by exon shuffling of families of human receptor domains, contain so-called A domains that exist as a series of multiple domains in several cell surface receptors (Silverman, J., et al, Nature Biotechnology (2005) 23, 1556-1561). AdNectins, derived from domains of human fibronectin, contain three loops that can be engineered for immunoglobulin-like binding to a target (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Similarly, tetranectins, derived from each human homotrimeric protein, contain loop regions in C-type lectin domains that can be engineered for desired binding (ibid).Peptoids that can act as protein ligands are oligo(N-alkyl)glycines that differ from peptides in that the side chains are linked to the amide nitrogen rather than the α-carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have even higher cell permeability than peptides (see, for example, Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

[0066] Still further examples of suitable proteinaceous binding molecules are EGF-like domains, Kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, Gla domains, SRCR domains, Kunitz / bovine pancreatic trypsin inhibitor domains, tendamistat, Kazal-type serine protease inhibitor domains, Trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I repeats, LDL-receptor class A domains, Sushi domains, Link domains, thrombospondin type I domains, immunoglobulin domains or immunoglobulin-like domains (e.g., domain antibodies or camel heavy chain antibodies), C-type lectin domains, MAM domains, von Willebrand factor type A domains, somatomedin B domains, WAP-type four-disulfide core domains, F5 / 8C-type domains, hemopexin domains, SH2 domains, SH3 domains, laminin-type EGF-like domains, C2 domains, “Kappabodies” (see Ill. et al, Protein Eng (1997) 10, 949-57), so-called “minibodies” (see Martin et al, EMBO J (1994) 13, 5303-5309), diabodies (see Holliger et al, PNAS USA (1993) 90, 6444-6448), so-called “Janusins” (see Traunecker et al, EMBO J (1991) 10, 3655-3659 or Traunecker et al, Int J Cancer (1992) Suppl 7, 51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitin, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. An example of a nucleic acid molecule having an antibody-like function is an aptamer. Aptamers fold into defined three-dimensional motifs and exhibit high affinity for a given target structure.

[0067] As used herein, the term "nucleic acid molecule" refers to any nucleic acid in any possible conformation, such as single-stranded, double-stranded, or combinations thereof. Nucleic acids include, for example, DNA molecules, RNA molecules, nucleotide analogs or analogs of DNA or RNA generated using nucleic acid chemistry, locked nucleic acid molecules (LNAs), PNA molecules (supra), and tecto-RNA molecules (e.g., Liu, B., et al, J. Am. Chem. Soc. (2004) 126, 4076-4077). PNA molecules are synthetic nucleic acid analogs having a pseudopeptide backbone in which the phosphodiester backbone present in DNA or RNA is replaced by repeating units of short aliphatic moieties containing an amino terminus and a carboxy terminus and which form amide bonds in oligomers or polymers. LNA molecules have a modified RNA backbone with a methylene bridge between C4' and O2' that locks the furanose ring in an N-type conformation, providing higher duplex stability and nuclease resistance for each molecule. Unlike PNA molecules, LNA molecules have a charged backbone. DNA or RNA can be of genomic or synthetic origin and can be single-stranded or double-stranded. Such nucleic acids can be, for example, mRNA, cRNA, synthetic RNA, genomic DNA, cDNA, synthetic DNA, copolymers of DNA and RNA, oligonucleotides, and the like. Each nucleic acid can further contain unnatural nucleotide analogs and / or can be linked to an affinity tag or label.

[0068] The method according to the present invention can be carried out at any temperature at which the viability of the target cells is at least essentially unimpaired. When reference is made herein to conditions that are at least essentially not harmful, not disadvantageous, or at least essentially do not impair viability, the conditions are such that the percentage of target cells that can be recovered in a state having full viability is at least 70%, including at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5%. In some embodiments, the method according to the present invention is carried out at a temperature of about 20 °C or less, for example, about 14 °C or less, about 9 °C or less, or about 6 °C or less. Depending on the target cells to be isolated, the suitable temperature range can be about 2 °C to about 45 °C, including, for example, about 2 °C to about 40 °C, about 3 °C to about 35 °C, or about 4 °C to about 30 °C when an aqueous medium is used to contain the target cells. In some embodiments, the method according to the present invention is carried out at a constant temperature value, or a selected temperature value ± about 5 °C, ± about 4 °C, ± about 3 °C, ± about 2 °C, ± about 1 °C, or ± about 0.5 °C. The temperature can be selected, for example, to have a value of about 5 °C, about 10 °C, about 15 °C, about 20 °C, or about 25 °C. In some embodiments, the temperature is changed during the method according to the present invention, i.e., increased, decreased, or varied by a combination thereof. The temperature can be changed, for example, within a range as defined above, for example, within a range of about 2 °C to about 40 °C or within a range of about 3 °C to about 35 °C. One skilled in the art can empirically determine the suitable temperature considering the nature of the cells and the isolation conditions. For example, temperature-insensitive cells such as cancer cells can be isolated even at high temperatures such as room temperature or 37 °C.

[0069] This method can also be performed using, for example, a kit of parts designed to perform the methods as detailed above. The kit may comprise a receptor binding reagent as defined above. The kit may comprise, for example, a receptacle filled with a receptor binding reagent, for example, a receptor binding reagent in the form of a solution. The kit may also comprise a chromatography matrix as defined above, for example, a cartridge, which may be pre-packed in a column. Associated with such a chromatography matrix and / or receptacle, in some embodiments, a warning in the form of instructions on how to use the kit for performing the method according to the invention is provided.

[0070] The present invention also provides the use of streptavidin, streptavidin mutein (analog), avidin, avidin mutein (analog), or mixtures thereof for isolating target cells via chromatography, which chromatography is gel filtration chromatography. For this purpose, in an embodiment, streptavidin, streptavidin mutein, avidin, avidin mutein, or any mixture thereof, for example, a mixture of both streptavidin and streptavidin mutein, is immobilized as an affinity reagent on the stationary phase of a "removal cartridge" as disclosed herein. As used herein, the term "streptavidin" includes wild-type streptavidin, streptavidin mutein, and streptavidin-like polypeptides. Similarly, as used herein, the term "avidin" includes wild-type avidin, as well as muteins of avidin, for example, deglycosylated avidin containing modified arginine that exhibits a more neutral pI and is available as an alternative to native avidin, including neutravidin. Commercially available forms of deglycosylated neutral forms of avidin include, for example, "Extravidin" available through Sigma-Aldrich or "NeutrAvidin" available from Thermo Scientific or Invitrogen.

[0071] As the wild-type streptavidin (wt-streptavidin), the amino acid sequence disclosed by Argarana et al, Nucleic Acids Res. 14(1986)1871-1882 is referred to. Streptavidin mutein is a polypeptide that is distinguished from the sequence of wild-type streptavidin by substitution, deletion, or addition of one or more amino acids and retains the binding properties of wt-streptavidin. Streptavidin-like polypeptides and streptavidin muteins are essentially immunologically equivalent to wild-type streptavidin and, in particular, are polypeptides that can bind biotin, biotin derivatives, or biotin analogs with the same or different affinities as wt-streptavidin. Streptavidin-like polypeptides or streptavidin muteins may contain amino acids that are not part of wild-type streptavidin or may contain only a part of wild-type streptavidin. Since the host does not have the enzymes necessary to convert the polypeptide produced by the host into the structure of wild-type streptavidin, streptavidin-like polypeptides are also polypeptides that are not identical to wild-type streptavidin. The term "streptavidin" also includes streptavidin tetramers and streptavidin dimers, in particular, streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers, and streptavidin heterodimers. Each subunit usually has a binding site for biotin or a biotin analog or a streptavidin-binding peptide. Examples of streptavidin or streptavidin mutein are mentioned, for example, in WO86 / 02077, DE19641876A1, U.S. Patent No. 6,022,951, WO98 / 40396, or WO96 / 24606.

[0072] In a preferred embodiment, the streptavidin mutein used for isolating target cells via chromatography, which is gel filtration chromatography, is the streptavidin mutein described in U.S. Patent No. 6,103,493 and DE19641876.3. These streptavidin muteins have at least one mutation in the region of amino acid positions 44 to 53 based on the amino acid sequence of wild-type streptavidin. Preferably, the N-terminus starts from the region of amino acids 10 to 16 of wild-type streptavidin and the C-terminus ends at the region of amino acids 133 to 142 of wild-type streptavidin. Examples of such preferred streptavidin muteins have a hydrophobic aliphatic amino acid instead of Glu at position 44, any amino acid at position 45, a hydrophobic aliphatic amino acid at position 46, and / or a basic amino acid instead of Val at position 47. The streptavidin mutein can be mutein Val44-Thr45-Ala46-Arg47 or streptavidin mutein (analog) Ile44-Gly45-Ala46-Arg47, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®.

[0073] The present invention also provides an apparatus for purifying target cells, the apparatus comprising at least one arrangement of the first and second stationary phases for chromatography as described above, meaning a chromatography column (selection cartridge) for selecting cells and a second chromatography column (removal cartridge) for removing reagents for isolating or staining the target cells. By performing such a two-step isolation procedure, target cells can be obtained that can be directly fed into the next desired application or selection cycle. In contrast to FACS and MACS selection, in the chromatography selection method of the present invention, no additional procedures such as washing and centrifugation are required between two selection cycles, and the cells are not functionally impaired by bound isolation reagents such as receptor-binding reagents or magnetic beads. Thus, the present invention provides, for the first time, a reliable, easy-to-construct, yet effective apparatus for target cell purification.

[0074] In accordance with the above, the apparatus of the claims of the present invention may comprise a plurality of arrangements of a first and a second stationary phase (chromatography column) fluidly connected in series. The apparatus may comprise a sample inlet fluidly connected to the first stationary phase of the first arrangement of the first and second stationary phases for chromatography. The apparatus may also comprise a sample outlet for the purified target cells, the sample outlet being fluidly connected to the second stationary phase of the last arrangement of at least one of the first and second stationary phases for chromatography. The apparatus may also comprise a competing reagent container fluidly connected to at least one of the first stationary phases of the arrangements of the first and second stationary phases for chromatography.

[0075] As will be readily understood by those skilled in the art from the disclosure of the present invention, other substance compositions, means, uses, methods, or processes existing or later developed that perform substantially the same function or produce substantially the same result as the corresponding exemplary embodiments described herein may also be used in accordance with the present invention.

Example

[0076] Experimental Example In this example, recombinantly produced Fab fragments against cell surface markers are used as receptor binding reagents. The Fab fragments are recombinantly expressed in E. coli or other hosts and contain a streptavidin binding peptide as binding partner C. Tetrameric streptavidin or tetrameric streptavidin mutein provides one or more binding sites Z. The Fab fragment is bound to streptavidin, and the streptavidin itself is covalently linked to beads. These beads are used for affinity column chromatography of a cell suspension containing a subpopulation of cells having an extracellular protein (receptor molecule) to which the Fab fragment can bind. Unbound cells are washed through this "selection cartridge", while bound cells are subsequently eluted with a biotin-containing buffer that disrupts the binding between the streptavidin binding peptide of the Fab fragment (acting as a receptor binding reagent) and the streptavidin mutein. As a result, the Fab fragment bound to the cell is released from the column, and due to the absence of the avidity effect, the Fab fragment dissociates from the target cell. Here, the suspension can be purified from residual Fab fragments and biotin by a second column chromatography (removal cartridge) using another gel (chromatography) matrix covalently bound to streptavidin, whereby the cells elute in the void volume and the Fab fragments and biotin bind quantitatively to the chromatography matrix. Here, the cells can be subjected to further purification cycles using different Fab fragments or any other receptor binding reagent in a similar manner. The column with the Fab fragment (selection cartridge) as well as the subsequent column for Fab and biotin removal (removal cartridge) can be combined in a continuous manner by simply arranging the columns linearly one after the other. By doing so, an automated cell purification system as shown in FIG. 6 can be provided by the present invention, enabling rapid, easy and cost-effective purification of target cells without the lack of magnetic beads or any manual interference.

[0077] This procedure enables, for example, the sequential purification of T cells starting from CD4+ purification and followed by CD25+ purification from the CD8+ fraction, resulting in a highly enriched fraction of regulatory T cells as shown below. Naturally, additional purification cycles using different Fab fragments are also possible.

[0078] Isolation of CD8+ T cells from human blood (typical one-step purification) Materials and methods Human blood was used to isolate PCMB by standard procedures.

[0079] Example 1: One-step purification of CD8+ cells via column chromatography Sephadex G50 (Sigma) was used as the stationary phase and covalently coupled to Strep-tactin® (recombinant streptavidin variant, IBA GmbH, Germany) using the CNBr method. A 50% suspension of Sephadex G50 contained 70 micrograms of covalently coupled Strep-tactin® / ml bead suspension. Strep-tactin® served as the affinity reagent immobilized on the affinity matrix prior to addition of the sample containing the receptor-binding reagent and target cells. A CD8+-binding Fab fragment in which the heavy chain was fused at the carboxy terminus to the tandem arrangement of two streptavidin-binding modules (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK, commercially available from IBA GmbH, Göttingen, Germany (catalog number: 6-8003)) was used as the (monovalent) receptor-binding reagent, and the streptavidin-binding peptide served as binding partner C.

[0080] To bind the Fab fragment to the CD8+ target cells, a 2 ml suspension of Sephadex G50 containing Strep-tactin® was incubated with 10 micrograms of the CD8+-binding Fab fragment at 4 °C for 20 minutes. The suspension was then filled into a plastic minicolumn (Mobitec, Göttingen, Germany) containing a 90 micrometer frit at the bottom. Thus, this column acts as a selection cartridge as defined herein. The column was equilibrated with PBS (phosphate-buffered saline) (PBSA buffer) containing 0.5% bovine serum albumin to obtain a bed volume of 1 ml. Then, 5 million cells from PCMB in 1 ml of PBSA were loaded onto the column to pass the sample through its affinity chromatography matrix. The column was then washed with 12 ml of PBSA. The wash buffer was collected and centrifuged at 3000 g for 6 minutes to pellet the cells washed from the column (pellet 1). Thereafter, 6 ml of PBSA containing 0.1 millimolar Biotin (Sigma) as a competing reagent was added to the column to elute the CD8+ cells reversibly immobilized on the column via the receptor-binding agent and the multimerizing agent. The fraction containing biotin was collected and centrifuged as above to pellet the cells (pellet 2).

[0081] The pellets from both fractions were resuspended in 1 ml of PBSA buffer for analysis.

[0082] Pellet 1 contained approximately 3.9 million cells and pellet 2 contained 0.7 million cells.

[0083] FACS analysis (data not shown) showed that compared to pellet 1, the CD8+ cells were greatly reduced (approximately 70%) from the starting material, and pellet 2 showed CD8+ cells with a purity of 68%. Thus, CD8+ target cells can be isolated via reversible immobilization / affinity chromatography.

[0084] This result was confirmed by the following experiment on the enrichment of CD8+ cells from PBMC, the results of which are shown in Figure 5. Enrichment was performed in two columns, both of which contained Sephadex-50 resin conjugated with Strep-Tactin® as described above. The first column (selection cartridge) (Diagrams B - D) was loaded with the CD8-binding Fab fragment commercially available from IBA GmbH described above. The second column (Diagrams E - G) served as a negative control for this selection cartridge (not to be confused with the removal cartridge, the "second column" placed after the selection cartridge) and was not loaded with this CD8-binding Fab fragment. Thus, the first column should show CD8-Fab specific cell enrichment, while the second column (negative control) should not. To measure the enrichment, the cell population of CD8+ T cells in PBMC was measured before starting the selection procedure (Figure 5, Diagram A, CD8+ T cells 18.4%, upper right quadrant). After applying the PBMC fraction onto the first column, the flow-through of cells that were not retained was measured (Diagram B, CD8+ T cells 6.3%). 12.1% (18.4% - 6.3%) or 66% of the CD8+ T cells bound to the column. These cells could then be specifically eluted by adding biotin buffer to disrupt the Strep-tag / Strep-Tactin® interaction and subsequent washing steps (Diagrams C+D, CD8+ T cells 47% and 62.2%). In contrast, the CD8+ T cell populations in the flow-through fraction (Diagram E, CD8+ T cells 17.0%) and the eluate fractions (Diagrams F and G) did not differ significantly from those measured before the selection procedure (Diagram A), so no enrichment of CD8+ T cells was seen in the second column (Diagrams D - E - F - G). A difference of approximately 1% of the applied cells accounts for cells that bound non-specifically to the column, indicating that 95% of the applied PBMC passed through the column.

[0085] Example 2: Removal of Biotin and Fab from C8+ Cells CD8+ cells were isolated as described above, except that the cells (6 ml of buffer) after biotin elution were passed directly through a column (6 ml bed volume) of Superflow™ Sepharose® beads to which Strep-Tactin® (IBA GmbH, Göttingen, Germany) was covalently attached with a binding capacity of 300 nmol biotin / ml. The Superflow™ Sepharose® beads serve as a gel permeation matrix for isolating / concentrating target cells, while the Strep-Tactin® immobilized on the beads has binding affinity for both biotin and the CD8+ Fab fragment bearing a streptavidin-binding peptide. Thus, Strep-Tactin® served as an affinity / removal reagent for biotin and the CD8+ Fab fragment. The eluate (6 ml) of this second column (which acted as a removal cartridge) filled with Superflow beads was collected.

[0086] Biotin and the Fab fragment were not detectable in the eluate containing the target cells when using a biotin assay and a Fab fragment assay using Western blotting (results not shown). Similar experiments were performed using FITC-labeled biotin (Sigma) and fluorescently labeled CD8+ Fab (IBA GmbH). The fact that this final eluate contained no Fab fragment or biotin was confirmed when measured using a high-sensitivity fluorometer. Thus, it was found that biotin and Fab were completely removed, while the eluate after Superflow™ / Strep-Tactin® chromatography contained 95% - 100% CD8+ cells and no obvious loss of cells was seen.

[0087] Example 3: Continuous purification in "linear flow" chromatography The final fractions, as described in Experiment 2, were free of biotin and Fab (both of which can interfere with subsequent purification procedures by blocking the Fab binding sites on Strep-Tactin®), so the purified CD8+ cells can proceed to another purification cycle, for example, using a CD25+ Fab fragment (or any other receptor molecule present on the surface of the isolated CD8+ target cells). Such sequential purification of T cells can be performed, for example, using the apparatus depicted in FIGS. 6a or 6b.

[0088] Example 4: Purification of Cells by Chromatography in a Planar Matrix (Nitrocellulose Membrane Coated with Strep-Tactin®) In this experiment, the ( "3-dimensional") column chromatography matrix (beads coated with Strep-Tactin®) used to purify cells in Examples 1 and 2 was replaced with a planar matrix coated with Strep-Tactin®. Experimental Procedure: 1) Non-covalent Attachment of Strep-Tactin® to the Nitrocellulose Membrane and Purification of CD8+ T Cells For the non-covalent attachment of Strep-Tactin® to the membrane, nitrocellulose membrane strips (24 cm 2, Whatman, UK) was placed in a Petri dish and incubated with 4 mg of Strep - tactin® (IBA GmbH) in 10 ml of PBS for 10 minutes, then washed 5 times with 20 ml of PBS. Then, 5 micrograms of CD8+ Fab fragment (catalog number: 6 - 8003 - 005, IBA GmbH, Gottingen, supra) was added to 5 ml of PBS and incubated at 4°C for 5 minutes. Then, 5 million cells (PBMC) in FACS buffer (0.5% BSA (w / v) in PBS pH 7.4) were added and incubated at 4°C for 10 minutes. Then, the membrane was washed 5 times with 10 ml of FACS buffer and the wash fractions were collected for FACS analysis. Then, the membrane was incubated in 10 ml of FACS buffer containing 1 mmol of biotin for 5 minutes. The resulting fraction was collected for FACS analysis.

[0089] This FACS analysis showed that the biotin - containing fraction was 99.1% pure with respect to CD8+ T cells. The yield of CD8+ cells was approximately 1.5% of the starting material. Thus, this experiment shows that target cells can be efficiently isolated by using planar chromatography in a "batch - like" manner.

[0090] Example 5: One - step purification of human CD8+ cells via column chromatography using Superflow® Agarose 3 ml of Superflow Strep-Tactin® (300 nanomoles of biotin binding, IBA GmbH, Gottingen, Germany)) was loaded onto a mini-column (Mobitec, Gottingen, Germany). The column was equilibrated with buffer (PBS + 0.5% bovine serum albumin, "FACS buffer"), and then loaded with PBMCs derived from human blood (10 million cells in 0.2 ml of FACS buffer) that had been pre-incubated with 12 micrograms of Fab against CD8 (catalog number: 6-8003, IBA GmbH, Gottingen) (as described above, the heavy chain of this Fab fragment is, at the carboxy terminus, fused to two streptavidin binding modules TIFF0007710427000004.tif5128 in a continuous arrangement and fused. This column acts as a selection cartridge as defined herein. The column was washed with 12 ml of FACS buffer by gravity flow and then eluted with 12 ml of FACS buffer containing 1 mM biotin.

[0091] The wash fraction (12 ml) of the FACS buffer contained only 1.77% CD8+ cells compared to 7.98% CD8+ cells in the starting fraction (A), so 77.8% of the CD8+ cells were retarded by the column. Elution with the biotin-containing buffer fraction (B, 12 ml) yielded approximately 65% of the bound CD8+ cells with a purity of 98.5%. This experiment also shows that CD8+ target cells can be isolated via reversible immobilization / affinity chromatography as described herein using commercially available chromatography matrices.

[0092] Example 6: One-step purification of human CD8+ cells via column chromatography Human CD8+ cells were isolated from density gradient (Ficoll) purified PBMCs by using a column prepared from 500 μl of Strep-Tactin®-agarose beads resin (cross-linked agarose with a smaller exclusion size compared to Superflow® Agarose, obtained from Agarose Beads Technologies, Madrid, Spain) functionalized with 10 μg of anti-CD8 Fab fragment (catalog number: 6-8003, IBA GmbH, Göttingen). For this purpose, a Fab fragment having a continuous arrangement of TIFF0007710427000005.tif5128 at the C-terminus of the heavy chain was loaded (immobilized) onto the Strep-Tactin®-agarose matrix by pumping 1000 μl of Fab-containing wash buffer (PBS + 0.5% bovine serum albumin) onto the column at a rate of 300 μl / min before cell purification. To purify the target cells, 1 × 10 freshly prepared PBMCs (in 2 ml of wash buffer) were automatically loaded onto the column using a peristaltic pump at a flow rate of 300 μl / min. Subsequently, unbound (CD8-negative) cells were removed from the column by repeated wash cycles (4×) at a rate of 2 ml / min with a total of 7 ml of wash buffer. Finally, the CD8+ target cells were eluted from the column by adding 5 ml of 100 μM D-biotin solution (V = 600 μl / min) and eluting at 2 ml / min with 5 ml of wash buffer to remove the bound cells from the affinity matrix. The resulting CD8-positive and -negative fractions were analyzed by flow cytometry. The CD8+ target cells were purified with a yield of 80% and a purity of 88%. The dot plots of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield of a representative selection, are shown in Figure 7. 8 Individuals of freshly prepared PBMCs (in 2 ml of wash buffer) were automatically loaded onto the column using a peristaltic pump at a flow rate of 300 μl / min. Subsequently, unbound (CD8-negative) cells were removed from the column by repeated wash cycles (4×) at a rate of 2 ml / min with a total of 7 ml of wash buffer. Finally, the CD8+ target cells were eluted from the column by adding 5 ml of 100 μM D-biotin solution (V = 600 μl / min) and eluting at 2 ml / min with 5 ml of wash buffer to remove the bound cells from the affinity matrix. The resulting CD8-positive and -negative fractions were analyzed by flow cytometry. The CD8+ target cells were purified with a yield of 80% and a purity of 88%. The dot plots of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield of a representative selection, are shown in Figure 7.

[0093] Example 7: One-step purification of human CD8+ cells via column chromatography from whole blood Human CD8+ cells were purified from whole blood by using a column prepared from 1200 μl of Strep-Tactin®-agarose beads resin (a cross-linked agarose with a smaller exclusion size compared to Superflow® agarose, obtained from Agarose Beads Technologies, Madrid, Spain) functionalized with 30 μg of anti-CD8 Fab fragment (catalog number: 6-8003, IBA GmbH, Göttingen). For this purpose, a Fab fragment having a continuous arrangement of TIFF0007710427000006.tif5128 at the C-terminus of the heavy chain was immobilized on the Strep-Tactin®-agarose matrix by pumping 1500 μl of a washing buffer containing the Fab fragment (PBS + 0.5% bovine serum albumin) onto the column at a rate of 300 μl / min before cell purification. To purify the target cells, 10 ml of freshly collected whole blood (diluted 1:1 with the washing buffer) was automatically loaded onto the column using a peristaltic pump at a flow rate of 300 μl / min. Subsequently, unbound (CD8-negative) cells were removed from the column by repeated washing cycles (4×) at a rate of 2 ml / min with a total of 13 ml of the washing buffer. Finally, the CD8+ target cells were eluted from the column by adding 10 ml of a 100 μM D-biotin solution (V = 600 μl / min) and eluting at 2 ml / min with 10 ml of the washing buffer to remove the bound cells from the affinity matrix. The resulting CD8-positive and -negative fractions were analyzed by flow cytometry. The CD8+ target cells were purified with a yield of 80% and a purity of 88%. The dot plots of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield for a representative selection, are shown in Figure 8. TIFF0007710427000006.tif5128 at the C-terminus of the heavy chain was immobilized on the Strep-Tactin®-agarose matrix by pumping 1500 μl of a washing buffer containing the Fab fragment (PBS + 0.5% bovine serum albumin) onto the column at a rate of 300 μl / min before cell purification. To purify the target cells, 10 ml of freshly collected whole blood (diluted 1:1 with the washing buffer) was automatically loaded onto the column using a peristaltic pump at a flow rate of 300 μl / min. Subsequently, unbound (CD8-negative) cells were removed from the column by repeated washing cycles (4×) at a rate of 2 ml / min with a total of 13 ml of the washing buffer. Finally, the CD8+ target cells were eluted from the column by adding 10 ml of a 100 μM D-biotin solution (V = 600 μl / min) and eluting at 2 ml / min with 10 ml of the washing buffer to remove the bound cells from the affinity matrix. The resulting CD8-positive and -negative fractions were analyzed by flow cytometry. The CD8+ target cells were purified with a yield of 80% and a purity of 88%. The dot plots of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield for a representative selection, are shown in Figure 8.

[0094] Example 8: Pipette-Based One-Step Purification of Mouse CD4+ Cells from Spleen Cells CD4+ cells were isolated from splenocytes by using a pipette tip loaded with 80 μl of Strep-Tactin®-agarose beads resin (cross-linked agarose with a smaller exclusion size compared to Superflow™ agarose, obtained from Agarose Beads Technologies, Madrid, Spain) functionalized with 2 μg of anti-CD4 Fab fragment. The pipette tip was filled with an agarose material by Phynexus Inc., USA. The Fab fragment used had a continuous array of TIFF0007710427000007.tif5128 at the C-terminus of the heavy chain and contained the wild-type variable domains of the CD4-binding antibody GK1.5 (Dialynas DP et al., Immunol Rev. 1983;74:29-56, GenBank Entry kappa light chain: M84148.1 GenBank Entry heavy chain: M84149.1). Prior to cell purification, loading / immobilization of the Fab fragment onto the Streptactin®-agarose matrix was performed by pipetting 400 μl of Fab fragment-containing wash buffer (PBS + 0.5% bovine serum albumin) onto the agarose chromatography matrix at a rate of 300 μl / min using a handheld electronic pipette. To purify the target cells, 1×10 The Fab fragment used had a continuous array of TIFF0007710427000007.tif5128 at the C-terminus of the heavy chain and contained the wild-type variable domains of the CD4-binding antibody GK1.5 (Dialynas DP et al., Immunol Rev. 1983;74:29-56, GenBank Entry kappa light chain: M84148.1 GenBank Entry heavy chain: M84149.1). Prior to cell purification, loading / immobilization of the Fab fragment onto the Streptactin®-agarose matrix was performed by pipetting 400 μl of Fab fragment-containing wash buffer (PBS + 0.5% bovine serum albumin) onto the agarose chromatography matrix at a rate of 300 μl / min using a handheld electronic pipette. To purify the target cells, 1×10 7Individual mouse spleen cells (in 0.5 ml of wash buffer) were applied onto the chromatography matrix present in the chip by means of a sample up-and-down cycle repeated three times using a pipette at a rate of 300 μl / min. This “batch-like” chromatography procedure of moving the buffer containing the cells up and down is equivalent to a procedure using a flow-based method to immobilize the cells onto the chromatography matrix. Subsequently, unbound (CD4-negative) cells were removed from the chip by washing three times repeatedly at a rate of 2 ml / min with 1 ml of wash buffer (by pipetting the wash buffer up and down). Finally, 1 ml of 100 μM D-biotin solution was added (V = 600 μl / min), and CD4+ target cells were eluted from the chip by removing the bound cells from the affinity matrix by eluting at a flow rate of 2 ml / min with 2 ml (2 × 1 ml) of wash buffer. The obtained CD4-positive and -negative fractions were analyzed by flow cytometry. CD8+ target cells were purified with a 95% yield and 85% purity. Dot plots of each of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield of representative selections, are shown in FIG. 9.

[0095] Example 9: One-step purification of human CD4+ cells via column chromatography Human CD4+ cells were isolated from density gradient (Ficoll) purified PBMCs using a pipette tip loaded with 80 μl of Strep-Tactin®-agarose beads resin (a cross-linked agarose with a smaller exclusion size compared to Superflow® Agarose obtained from Agarose Beads Technologies, Madrid, Spain) functionalized with 2 μg of anti-CD4 Fab fragment. The CD4 Fab fragment used was a mutant of the 13B8.2 Fab fragment described in U.S. Patent No. 7,482,000 and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003)). The mutant Fab fragment, called "m13B8.2", has the variable domain of the CD4-binding murine antibody 13B8.2, as well as a constant domain consisting of a gamma1 type (type gamma1) constant human CH1 domain and a kappa type constant human light chain domain for the heavy chain. Compared to the variable domain of the 13B8.2 Fab fragment in m13B8.2, the His residue at position 91 of the light chain (position 93 in SEQ ID NO: 2) is mutated to Ala, and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO: 1) is mutated to Ala. Furthermore, the Fab fragment m13B8.2 has a tandem arrangement of two streptavidin-binding modules TIFF0007710427000008.tif6128 at the C-terminus of the heavy chain. The Fab fragment was immobilized on the Strep-Tactin®-agarose matrix by pipetting 200 μl of Fab fragment-containing wash buffer at a rate of 300 μl / min using a handheld electronic pipette prior to cell purification. To select target cells, 1×10 7Individual freshly prepared PBMCs (in 0.5 ml wash buffer) (PBS + 0.5% bovine serum albumin) were automatically applied onto the chromatography matrix present in the chip by means of a sample up-and-down cycle repeated three times using a pipette at a rate of 300 μl / min. Subsequently, unbound (CD4-negative) cells were removed from the chip by washing three times in succession at a rate of 2 ml / min with 1 ml of wash buffer (by pipetting the wash buffer up and down). Finally, 1 ml of 100 μM D-biotin solution was added (V = 600 μl / min), and the CD4+ target cells were eluted from the chip by removing the bound cells from the affinity matrix by eluting at a flow rate of 2 ml / min with 2 ml (2 × 1 ml) of wash buffer. The resulting CD4-positive and -negative fractions were analyzed by flow cytometry. The CD4+ target cells were purified with a yield of 90% and a purity of 99%. The dot plots of each of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield of a representative selection, are shown in Figure 10.

[0096] Example 10: Pipette-Based One-Step Purification of Human CD4+ Cells from Whole Blood CD4+ cells were isolated from whole blood by using a pipette tip loaded with 80 μl of Strep-Tactin®-agarose beads resin (a cross-linked agarose with a smaller exclusion size compared to Superflow™ Agarose, obtained from Agarose Beads Technologies, Madrid, Spain) functionalized with 0.5 μg of anti-CD4 Fab fragment. The CD4-binding Fab fragment m13B8.2 used in Example 9 was also used in Example 10. The Fab fragment was immobilized on the Strep-Tactin®-agarose matrix by pipetting 200 μl of Fab-containing wash buffer at a rate of 300 μl / min using a handheld electronic pipette prior to cell isolation. To isolate the target cells, 2 ml of freshly collected whole blood (diluted 1:1 with wash buffer) (PBS + 0.5% bovine serum albumin) was automatically applied onto the chromatography matrix present in the tip by an up-and-down cycle repeated 3 times using a pipette at a rate of 300 μl / min. Subsequently, unbound (CD4-negative) cells were removed from the tip by washing 5 times repeatedly at a rate of 2 ml / min with 1 ml of wash buffer (by pipetting up and down). Finally, 1 ml of 100 μM D-biotin solution was added (V = 600 μl / min), and the CD4+ target cells were eluted from the tip by removing the bound cells from the affinity matrix by eluting with 2 ml (2 × 1 ml) of wash buffer at 2 ml / min. The resulting CD4-positive and -negative fractions were analyzed by flow cytometry. The CD4+ target cells were purified with a yield of 88% and a purity of 70%. The dot plots of the starting fraction, negative fraction, and positive fraction, as well as the corresponding purity and yield of a representative selection, are shown in FIG. 11.

[0097] In this context, it should be noted that further purification or further use of the target cells obtained in Examples 4 to 11, biotin as the eluent, and the Fab fragments as the respective receptor-binding reagents can be removed from the target cell sample using the "removal cartridge" as described in Example 2.

[0098] A listing or description of previously published documents herein is not necessarily to be construed as an admission that such document is part of the state of the art or common general knowledge.

[0099] The invention illustratively described herein can be suitably practiced in the absence of any element not explicitly disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively without limitation. Further, the terms and expressions used herein are used as terms of description and not of limitation, and are not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the invention has been clearly disclosed by way of example and optional features, modifications and variations of the invention disclosed and embodied herein may be resorted to by those skilled in the art, and such modifications and variations are to be understood as being within the scope of the invention.

[0100] The invention has been generally described herein in broad terms. Each of the narrower species and subgeneric classifications falling within the scope of that general disclosure also forms part of the invention. This includes the general description of the invention with any conditions or negative limitations removing any subject matter from that genus, whether or not the material being removed is explicitly recited herein.

[0101] Other aspects are within the scope of the following claims. Further, when features or aspects of the invention are described in terms of Markush groups, one of ordinary skill in the art will recognize that the invention is also described with respect to any individual member or subgroup of members of that Markush group.

[0102] Sequence information SEQUENCE LISTING <110> JUNO THERAPEUTICS GMBH <120> Chromatographic isolation of cells and other complex biological materials <150> US 61 / 602,150 <151> 2012-02-23 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 253 <212> PRT <213> human <220> <223> Heavy Chain of Fab Fragment m13B8.2 <400> 1 Ala Met Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro 1 5 10 15 Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr 20 25 30 Thr Phe Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Gly Val Ile Trp Ala Ser Gly Ile Thr Asp Tyr Asn Val Pro 50 55 60 Phe Met Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val 65 70 75 80 Phe Phe Lys Leu Asn Ser Leu Gln Pro Asp Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Lys Asn Asp Pro Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Gly Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Ser 210 215 220 Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 245 250 <210> 2 <211> 218 <212> PRT <213> human <220> <223> Light Chain of Fab Fragment m13B8.2 <400> 2 Ala Met Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Glu Thr Val Thr Phe Thr Cys Arg Ala Ser Glu Met Ile Tyr 20 25 30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu 35 40 45 Leu Val His Asp Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Gly Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Thr Leu 65 70 75 80 Gln Pro Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Ala His Tyr Gly Asn 85 90 95 Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ile 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys Gly Ser 210 215 <210> 3 <211> 8 <212> PRT <213> artificial <220> <223> streptavidin-binding peptide <400> 3 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 4 <211> 9 <212> PRT <213> artificial <220> <223> HA-tag <400> 4 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 5 <211> 11 <212> PRT <213> artificial <220> <223> VSV-G.tag <400> 5 Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 1 5 10 <210> 6 <211> 11 <212> PRT <213> artificial <220> <223> HSV-tag <400> 6 Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp 1 5 10 <210> 7 <211> 10 <212> PRT <213> artificial <220> <223> T7 epitope <400> 7 Ala Ser Met Thr Gly Gly Gln Gln Met Gly 1 5 10 <210> 8 <211> 10 <212> PRT <213> artificial <220> <223> myc-epitope <400> 8 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 9 <211> 14 <212> PRT <213> artificial <220> <223> V5-tag <400> 9 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 10 <211> 11 <212> PRT <213> artificial <220> <223> MAT-tag <400> 10 His Asn His Arg His Lys His Gly Gly Gly Cys 1 5 10 <210> 11 <211> 4 <212> PRT <213> artificial <220> <223> streptavidin mutein <400> 11 Val Thr Ala Arg 1 <210> 12 <211> 4 <212> PRT <213> artificial <220> <223> streptavidin mutein analog <400> 12 Ile Gly Ala Arg 1 <210> 13 <211> 30 <212> PRT <213> artificial <220> <223> streptavidin binding module <400> 13 Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 20 25 30

Claims

Claim 1 A method for isolating a target cell, wherein the target cell has a receptor molecule on the surface of the target cell, and the method comprises: providing a sample containing the target cell; providing a receptor-binding reagent comprising a binding site B and a binding partner C; The binding site B contained in the receptor-binding reagent can specifically bind to a receptor molecule on the surface of a target cell, and has a dissociation constant (K D ) is about 10 -3 ~about 10 -7 K within M D or the dissociation rate constant (koff) for the binding between the receptor-binding reagent and the receptor molecule via the binding site B is about 3×10 -5 sec -1 or greater, a binding partner C contained in said receptor-binding reagent being capable of reversibly binding to a binding site Z of the affinity reagent; and subjecting the sample to chromatography on a suitable stationary phase, on which an affinity reagent is immobilized; wherein the affinity reagent comprises a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor-binding reagent, and the binding site B of the receptor-binding reagent binds to the receptor molecule on the surface of the target cell, whereby the target cell is reversibly immobilized on the stationary phase; a method comprising the above steps. Claim 2 The method according to claim 1, wherein the affinity reagent comprises two or more binding sites Z capable of reversibly binding to the binding partner C contained in the receptor-binding reagent. Claim 3 The method according to claim 1, further comprising the step of loading a competing reagent onto the stationary phase, wherein the competing agent is capable of disrupting the binding between the partner C of the receptor-binding reagent and the binding site Z of the affinity reagent, thereby replacing the receptor-binding agent. Claim 4 The method according to claim 3, wherein the competing reagent is capable of competitively binding to the binding site Z of the affinity reagent. Claim 5 The method according to any one of claims 1 to 4, wherein the receptor-binding reagent is immobilized on the stationary phase before applying the sample containing the target cell to the stationary phase. Claim 6 The method according to any one of claims 1 to 5, wherein the sample contains a mixture of the target cell and additional cells, the additional cells lack the receptor molecule on their cell surface, and the method comprises separating the target cell from the additional cells. Claim 7 A method for isolating a target cell, wherein the target cell has a receptor molecule on the surface of the target cell, and the method comprises: providing a sample, the sample containing the target cell and a receptor-binding reagent; wherein the receptor-binding reagent comprises a binding site B and a binding partner C, and the binding site B contained in the receptor-binding reagent is capable of specifically binding to the receptor molecule; and A step of subjecting the sample to chromatography using a suitable stationary phase, wherein the stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix contains an affinity reagent, and the affinity reagent contains a binding site Z that specifically binds to binding partner C contained in the receptor-binding reagent, whereby the target cells are isolated A method comprising the above step **Claim 8** The method according to any one of claims 1 to 7, wherein the chromatography is column chromatography or planar chromatography **Claim 9** The method according to claim 7, wherein the step of subjecting the sample to chromatography enables binding partner C contained in the receptor-binding reagent to form a complex with binding site Z of the affinity reagent of the stationary phase to which it specifically binds, whereby the receptor-binding reagent is immobilized on the stationary phase **Claim 10** The method according to claim 7 or 9, wherein binding partner C contained in the receptor-binding reagent can specifically bind to binding site Z of the affinity reagent, and the affinity reagent contains two or more binding sites Z that can specifically bind to binding partner C contained in the receptor-binding reagent **Claim 11** The method according to claim 9 or 10, wherein the affinity reagent is covalently immobilized on the affinity chromatography matrix and / or the gel filtration matrix **Claim 12** The method according to any one of claims 7 to 11, wherein the sample further contains a competing reagent, and the competing reagent binds to binding site Z of the affinity reagent that specifically binds to binding partner C contained in the receptor-binding reagent, whereby the competing reagent is immobilized on the stationary phase **Claim 13** Further comprising a step of forming a sample The step of forming the sample Comprises providing a source sample containing target cells, and Adding a receptor-binding reagent to the source sample The method according to any one of claims 1 to 4 **Claim 14** The method according to claim 13, wherein the source sample is expected to contain a plurality of target cells, and the source sample is contacted with a plurality of receptor-binding reagents, and an excess amount of receptor-binding reagent is provided for the expected number of target cells **Claim 15** The method according to any one of claims 1 to 14, wherein the sample is a fluid or contains a fluid.

16. The method according to any one of claims 1 to 15, wherein the sample contains a body fluid.

17. The method according to claim 16, wherein the body fluid is blood or a blood component.

18. The method according to any one of claims 1 to 17, wherein the step of subjecting the sample to chromatography comprises passing the sample through a stationary phase of a chromatography column and washing the stationary phase with a fluid mobile phase, and the fluid mobile phase is essentially free of a receptor-binding reagent.

19. The method according to any one of claims 1 to 18, wherein the step of subjecting the sample to chromatography comprises eluting target cells from a chromatography matrix.

20. The method according to claim 19, further comprising a step of recovering the target cells.

21. A method for chromatographically isolating target cells from a sample, wherein the target cells have receptor molecules on the surface of the target cells, and the method comprises: providing a sample containing the target cells; providing a receptor-binding reagent comprising a binding site B and a binding partner C, wherein the binding site B contained in the receptor-binding reagent can specifically bind to a receptor molecule on the surface of the target cells; wherein the binding partner C contained in the receptor-binding reagent can reversibly bind to a binding site Z of an affinity reagent; subjecting the sample to chromatography on a suitable stationary phase, on which an affinity reagent is immobilized, wherein the affinity reagent comprises a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor-binding reagent, and the binding site B of the receptor-binding reagent binds to a receptor molecule on the surface of the target cells, whereby the target cells are reversibly immobilized on the stationary phase; providing a competing reagent comprising a binding site that specifically binds to the binding site Z of the affinity reagent; loading the competing reagent onto a first stationary phase, thereby disrupting the non-covalent reversible complex formed between the (plural) receptor-binding reagent, the receptor molecule, and the affinity reagent; recovering an eluted sample from the eluate of the first stationary phase, the eluted sample containing the target cells. A step of subjecting the elution sample to chromatography on a suitable second stationary phase, wherein the second stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, and the gel filtration and / or affinity chromatography matrix contains an affinity reagent having a binding site Z that specifically binds to binding partner C contained in the receptor binding reagent, and A step of passing the elution sample through a second chromatography column A method comprising.

22. The method according to claim 21, wherein an affinity reagent is immobilized on an affinity chromatography matrix and / or a gel filtration matrix as the second stationary phase.

23. The method according to claim 21 or 22, wherein the step of passing the elution sample through the second stationary phase includes a step in which a competing reagent forms a complex with the binding site Z of the affinity reagent, so that the competing reagent is immobilized on the stationary phase of the second chromatography column.

24. The method according to any one of claims 21 to 23, wherein each of the first and second stationary phases is contained in a column or is a planar stationary phase.

25. The binding between the binding site B of the receptor-binding reagent and the receptor molecule has a dissociation constant (K -2 ), within the range of about 10 -10 M to about 10 D M, the method according to any one of claims 21 to 24.

26. The dissociation constant (K D ), with respect to the binding of the receptor-binding reagent and the receptor molecule via the binding site B, is of low affinity, or the dissociation rate constant (koff) with respect to the binding of the receptor-binding reagent and the receptor molecule via the binding site B is about 3×10 -5 sec -1 or greater, the method according to claim 25.

27. The reversible binding between the binding partner C of the receptor binding reagent and the binding site Z of the affinity reagent is about 10 -2 to about 10 -13 M dissociation constant (K D ), The method according to any one of claims 1 to 26.

28. The method according to any one of claims 1 to 27, wherein the stationary phase is a non-magnetic material or a non-magnetizable material.

29. The method according to claim 28, wherein the stationary phase includes or consists of one of a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, a polysaccharide grafted silica, a polyvinylpyrrolidone grafted silica, a polyethylene oxide grafted silica, a poly(2-hydroxyethyl aspartoamide) silica, a poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of acrylate or acrylamide and diol, a copolymer of polysaccharide and N,N'-methylenebisacrylamide, and any combination of two or more thereof.

30. The method according to any one of claims 1 to 29, wherein the affinity chromatography matrix and / or the gel filtration matrix includes or consists of a monolithic matrix, a granular matrix, or a planar matrix.

31. The method of claim 30, wherein the particulate matrix has an average particle size of from about 5 μm to about 200 μm or from about 5 μm to 600 μm or from about 5 μm to 1500 μm.

32. The method according to any one of claims 1 to 31, wherein the affinity chromatography matrix and / or the gel filtration matrix has an average pore size of from 0 to about 500 nm.

33. The method according to any one of claims 1 to 32, wherein the receptor binding reagent is selected from the group consisting of immunoglobulins, functional fragments of immunoglobulins, proteinaceous binding molecules having immunoglobulin-like functions, aptamers, and MHC molecules.

34. The method according to any one of claims 1 to 33, wherein the binding partner C comprised in the receptor binding reagent comprises one of biotin, a biotin analog, a streptavidin-binding peptide, and an avidin-binding peptide, and the affinity reagent comprises streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof.

35. The method according to any one of claims 1 to 34, wherein the target cell is a mammalian cell.

36. The method according to any one of claims 1 to 35, wherein the target cell is a cell having a cell nucleus.

37. The method of claim 36, wherein the target cell is a leukocyte or a stem cell.

38. The method of claim 37, wherein the leukocyte is a lymphocyte.

39. Use of a receptor-binding reagent and / or an affinity reagent for isolating target cells via chromatography using a stationary phase, wherein the target cells have receptor molecules on the surface of the target cells, the receptor-binding reagent comprises a binding site B and a binding partner C, the binding site of the receptor-binding reagent can specifically bind to the receptor molecule of the target cell, and the dissociation constant (K D for the binding of the receptor-binding reagent to the receptor molecule via the binding site B is of low affinity or the dissociation rate constant (koff) for the binding of the receptor-binding reagent to the receptor molecule via the binding site B is about 3×10 -5 sec -1 or greater, and the binding partner C comprised in the receptor-binding reagent can reversibly bind to the binding site Z of the affinity reagent.

40. Use according to claim 39, wherein the affinity reagent comprises two or more binding sites Z that reversibly bind to the binding partner C comprised in the receptor binding reagent.

41. Use according to claim 39 or 40, wherein the receptor binding reagent is one of an immunoglobulin, a functional fragment of an immunoglobulin, a proteinaceous binding molecule having immunoglobulin-like functions, an aptamer, and an MHC molecule.

42. Use according to any one of claims 39 to 41, wherein the binding partner comprised in the receptor binding reagent comprises one of biotin, a biotin analog, a streptavidin-binding peptide, and an avidin-binding peptide, and the affinity reagent comprises one of streptavidin, a streptavidin analog, avidin, and an avidin analog.

43. Use of streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof for isolating target cells via chromatography, wherein the chromatography is gel filtration chromatography.

44. The use according to claim 43, wherein the chromatography is column chromatography or planar chromatography.

45. The use according to claim 43 or 44, wherein the gel filtration chromatography is performed on a stationary phase having streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof immobilized thereon.

46. The use according to any one of claims 43 to 45, wherein the target cells are cells having a cell nucleus.

47. The use according to claim 46, wherein the cells having a cell nucleus are lymphocytes.

48. Use of a chromatographic matrix selected from a cellulose membrane, a plastic membrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel, a polysaccharide grafted silica, a polyvinylpyrrolidone grafted silica, a polyethylene oxide grafted silica, a poly(2-hydroxyethyl aspartamide) silica, a poly(N-isopropylacrylamide) grafted silica, a styrene-divinylbenzene gel, a copolymer of acrylate or acrylamide and diol, a copolymer of polysaccharide and N,N'-methylenebisacrylamide, and any combination of two or more thereof for separating cells containing a cell nucleus.

49. The use according to claim 48, wherein the cells containing a cell nucleus are lymphocytes.

50. The use according to claim 48 or 49, wherein the copolymer of polysaccharide and N,N'-methylenebisacrylamide is Sephacryl (registered trademark).

51. The use according to claim 48 or 49, wherein the polysaccharide gel is Sepharose (registered trademark).

52. The use according to claim 48 or 49, wherein the agarose gel is a cross-linked dextran gel such as Sephadex (registered trademark).

53. The use according to claim 48 or 49, wherein the copolymer of acrylate and diol is Toyopearl (registered trademark).

54. Use according to claim 48 or 49, wherein the polyacrylamide gel is one of Fractogel® and Bio-Gel®.

55. Use according to any one of claims 48 to 54, wherein the chromatography gel is contained in a column.

56. Use according to claim 55, wherein the column is a cartridge.

57. A kit of parts for isolating a target cell, wherein the target cell has receptor molecules on the target cell surface, and the kit comprises (a) a receptor-binding reagent comprising a binding site B and a binding partner C, wherein the binding site B contained in the receptor-binding reagent can specifically bind to the receptor molecules on the surface of the target cell, and the binding partner C contained in the receptor-binding reagent can reversibly bind to a binding site Z on a multimerization reagent; and (b) a stationary phase suitable for cell separation, wherein the stationary phase is defined by a gel filtration matrix and / or an affinity chromatography matrix, and the affinity chromatography matrix or the gel filtration and affinity chromatography matrix comprises an affinity reagent having a binding site Z that can reversibly bind to the binding partner C contained in the receptor-binding reagent A kit comprising the above.

58. The kit of parts according to claim 57, wherein the stationary phase is contained in a chromatography column or is a planar stationary phase.

59. The kit of parts according to claim 57 or 58, further comprising a second stationary phase suitable for cell separation / target cell separation from other components in the sample, wherein the second stationary phase is defined by an affinity chromatography matrix, and the affinity chromatography matrix has an affinity reagent immobilized thereon, and the affinity reagent comprises a binding site Z that can specifically bind to the binding partner C contained in the receptor-binding reagent.

60. The kit of parts according to claim 59, wherein the second stationary phase is contained in a chromatography column or is a planar stationary phase.

61. A method for isolating a target cell, wherein the target cell has receptor molecules on the target cell surface, and the method comprises providing a sample containing the target cell. Providing a receptor-binding reagent comprising a monovalent binding site B and a binding partner C, wherein the receptor-binding reagent is selected from the group consisting of a monovalent antibody fragment, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule, The monovalent binding site B comprised in the receptor-binding reagent is capable of specifically binding to a receptor molecule on the surface of a target cell, and the binding partner C comprised in the receptor-binding reagent is capable of reversibly binding to the binding site Z of an affinity reagent, and Subjecting the sample to chromatography on a suitable stationary phase, the stationary phase having an affinity reagent immobilized thereon, the affinity reagent comprising a binding site Z, the binding site Z forming a reversible bond with the binding partner C comprised in the receptor-binding reagent, and the binding site B of the receptor-binding reagent binding to a receptor molecule on the surface of the target cell, whereby the target cell is reversibly immobilized on the stationary phase, A method comprising.

62. The method according to claim 61, wherein the monovalent antibody fragment is a Fab fragment, an Fv fragment, or a single-chain Fv fragment.

63. The method according to claim 61, wherein the proteinaceous binding molecule having an immunoglobulin-like function is selected from the group consisting of muteins based on polypeptides of the lipocalin family, globodies, proteins based on ankyrin skeletons, proteins based on crystallin skeletons, adnectins, and avimers.

64. Use of a receptor-binding reagent and / or an affinity reagent for isolating a target cell via chromatography using a stationary phase, the target cell having a receptor molecule on the surface of the target cell, The receptor-binding reagent is selected from the group consisting of a monovalent antibody fragment, a proteinaceous binding molecule having an immunoglobulin-like function, an aptamer, and an MHC molecule, The receptor-binding reagent comprises a binding site B and a binding partner C, and the binding site of the receptor-binding reagent is capable of specifically binding to the receptor molecule of the target cell, The binding partner C comprised in the receptor-binding reagent is capable of reversibly binding to the binding site Z of the affinity reagent.

65. An apparatus for purifying a target cell, comprising at least one arrangement of a first and a second stationary phase for chromatography, The first stationary phase is suitable for cell separation, and the first stationary phase is defined by an affinity chromatography matrix, on which an affinity reagent is immobilized. The affinity reagent has at least one binding site Z that can reversibly bind to binding partner C included in the receptor binding reagent. The second stationary phase is suitable for cell separation, and the second stationary phase is a gel filtration matrix and / or an affinity chromatography matrix, and the affinity chromatography matrix or the gel filtration and affinity chromatography matrix includes an affinity reagent having a binding site Z that specifically binds to binding partner C included in the receptor binding reagent.

66. The apparatus according to claim 65, wherein the affinity reagent immobilized on the affinity chromatography matrix of the first stationary phase includes two or more binding sites Z that can reversibly bind to binding partner C included in the receptor binding reagent.

67. The apparatus according to claim 65 or 66, wherein each of the first and second stationary phases is included in a chromatography column or is a planar stationary phase.

68. The apparatus according to any one of claims 65 to 67, including a plurality of first and second stationary phases.

69. The apparatus according to any one of claims 65 to 68, further comprising a plurality of arrangements of a plurality of first and second stationary phases fluidly connected in series.

70. The apparatus according to claim 69, comprising a sample inlet fluidly connected to the first stationary phase of the first arrangement of the first and second stationary phases for chromatography.

71. The apparatus according to claim 70, comprising a sample outlet for purified target cells, and the sample outlet is fluidly connected to the second stationary phase of the last arrangement of at least one of the first and second stationary phases for chromatography.

72. The apparatus according to any one of claims 65 to 71, comprising a competing reagent container fluidly connected to at least one of the first stationary phases of the arrangements of the first and second stationary phases for chromatography.

73. A method for screening target cells for recombinant expression of a desired receptor molecule on the surface of the target cells, wherein the desired receptor molecule is expressed on the surface of the target cells, and the method comprises A step of providing a sample, the sample comprising target cells that may recombinantly express the desired target receptor, A step of providing a receptor binding reagent comprising a binding site B and a binding partner C, The binding site B contained in the receptor binding reagent can specifically bind to the desired receptor molecule on the target cell surface, and the binding partner C contained in the receptor binding reagent can reversibly bind to the binding site Z of the affinity reagent, A step of subjecting the sample to chromatography on a suitable stationary phase, the stationary phase having an affinity reagent immobilized thereon, The affinity reagent comprises a binding site Z, the binding site Z forms a reversible bond with the binding partner C contained in the receptor binding reagent, and the binding site B of the receptor binding reagent binds to the receptor molecule on the target cell surface, whereby the target cell is reversibly immobilized on the stationary phase, A method comprising.

74. The screening method according to claim 73, wherein the desired receptor molecule is endogenous or exogenous to the target cell.

75. The screening method according to claim 73 or 74, wherein the target cell is transfected with a nucleic acid encoding the desired receptor molecule.

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