Chromatographic isolation of cells and other complex biological materials

A method of chromatographically isolating a target cell from a sample, wherein the target cell has a receptor molecule on the surface of the target cell, the method comprising:

• providing a sample comprising one or more target cells and a receptor binding reagent, the receptor binding reagent comprising (i) a binding site B capable of specifically binding to the receptor molecule on the target cell surface and (ii) a binding partner C capable of reversibly binding to a first affinity reagent, wherein the binding partner C comprises biotin, a biotin analogue, a streptavidin binding peptide or an avidin binding peptide,
• exposing the sample to chromatography on a first stationary phase, the first stationary phase having immobilized thereon a first affinity reagent comprising streptavidin, a streptavidin mutein, avidin, an avidin mutein or a mixture thereof, said first affinity reagent comprising a plurality of first binding site Z, wherein said first binding site Z is capable of forming a non-covalent reversible complex with the binding partner C and wherein the binding site B is capable of binding to the receptor molecule on the target cell surface, thereby reversibly immobilizing the one or more target cells on the stationary phase;
• loading a competition reagent onto the first stationary phase, the competition reagent comprising a binding site that is capable of specifically binding to the first binding site Z, wherein binding of the competition reagent to the first binding site Z allows disruption of the non-covalent reversible complex formed between the binding partner C of the receptor binding reagent and the first binding site Z of the affinity reagent;
• recovering an elution sample from an eluate of the first stationary phase, wherein the elution sample comprises the one or more target cells, the competition reagent, and the receptor binding reagent;
• exposing the elution sample to chromatography on a second stationary phase, the second stationary phase comprising a gel filtration matrix and/or affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix comprises a second affinity reagent comprising streptavidin, a streptavidin mutein, avidin, an avidin mutein or a mixture thereof, said second affinity reagent having a plurality of second binding site Z that specifically bind to the binding partner C in the receptor binding reagent, and
• passing the elution sample through the second stationary phase under conditions sufficient to allow binding of the second binding site Z with the binding partner C, thereby isolating at least one target cell that is free from a bound receptor binding reagent.

A method of isolating a target cell, wherein the target cell has a receptor molecule on the target cell surface, the method comprising:

• contacting a sample comprising one or more target cells with a receptor binding reagent, the receptor binding reagent comprising a monovalent binding site B and a binding partner C, wherein the binding partner C comprises biotin, a biotin analogue, a streptavidin binding peptide, or an avidin binding peptide, wherein:
• the receptor binding reagent is selected from the group consisting of a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like functions, an aptamer and an MHC molecule,
• the monovalent binding site B is capable of specifically binding to the receptor molecule on the target cell surface, wherein (i) the dissociation constant (KD) for the binding of the receptor binding reagent to the receptor molecule is in the range of about 10−3 to about 10−7 M and/or (ii) the binding of the receptor binding reagent to the receptor molecule has a koff rate of about 3×10−5 sec−1 or greater,
• the binding partner C is capable of reversibly binding to a binding site Z of an affinity reagent, wherein the affinity reagent comprises streptavidin, a streptavidin mutein, avidin, an avidin mutein or a mixture thereof, and
• exposing the sample to chromatography on a stationary phase, the stationary phase having the affinity reagent comprising the binding site Z immobilized thereon, wherein said binding site Z is capable of forming a non-covalent reversible complex with the binding partner C and wherein the binding site B is capable of binding to the receptor molecule on the target cell surface;
• thereby reversibly immobilizing the one or more target cells on the stationary phase.