Chromatographic isolation of cells and other complex biological materials
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Publication Number
US-12066365-B2
Publication Date
2024-08-20
Expiration Date
Abstract
The present invention relates to the chromatographic isolation of a target cell or another complex biological material, in particular by column chromatography such as affinity chromatography or gel permeation chromatography. The invention employs a receptor binding reagent that binds to a receptor molecule that is located on the surface of a target cell. The invention in general provides novel methods for the traceless isolation of biologic materials such as cells, cell organelles, viruses and the like. The invention also relates to an apparatus for the isolation of cells and other complex biological materials.
Core Innovation
The invention provides a chromatographic affinity/gel-filtration approach for isolating a target cell using a receptor binding reagent that binds a cell-surface receptor. The method uses selection affinity chromatography in which the receptor binding reagent is reversibly immobilized on a stationary phase through a second binding interaction between a binding partner C and an affinity reagent on the stationary phase at a binding site Z. In this arrangement, the binding site Z has a lower dissociation constant (KD) for biotin or a biotin analog than for the binding partner C, enabling immobilization of the receptor binding reagent on the stationary phase chromatographic matrix.
The receptor binding reagent comprises a Fab monomer fragment that binds CD4 on the surface of lymphocytes, and the reagent further includes a binding partner C that comprises a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment. The reversible immobilization forms a complex in the selection affinity chromatography column in which multimers of the monomeric Fab fusion bind to the target cells, thereby reversibly immobilizing the one or more CD4+ lymphocyte target cells on the chromatographic matrix. Unbound cells are removed by washing prior to elution.
Elution is achieved by contacting the selection affinity chromatography column with a competition reagent selected from biotin or a biotin analog capable to disrupt the binding between the streptavidin-binding peptide of the receptor binding reagent and the streptavidin mutein, thereby displacing the receptor binding reagent and releasing the CD4+ lymphocytes. The eluate comprises the one or more CD4+ lymphocyte target cells, the Fab monomers, and biotin or a biotin analog. The approach is further associated with a removal chromatography concept using size exclusion to separate/retain Fab and biotin components while isolating the target cells, and is described with serial selection/removal cartridges and formats including a planar (nitrocellulose) matrix and a pipette tip format.
From the provided description and claim context, the problem being solved is to isolate target cells while enabling reversible release without covalent cleavages and while reducing needs for removal steps, including reducing reliance on magnetic beads. The invention also aims to maintain compatible chromatographic separation using a nonmagnetic or non-magnetizable stationary phase material, where binding and release depend on reversibly formed non-covalent complexes between the receptor binding reagent and the immobilized affinity reagent.
Claims Coverage
The provided claims include one independent claim for isolating a target cell, specifically a CD4+ lymphocyte, using nonmagnetic chromatographic matrices with a streptavidin mutein affinity reagent and a reversible Fab/peptide/biotin competition elution scheme. The independent claim includes an explicit selection affinity chromatography column, washing, and competition-based elution, with optional refinement appearing in dependent claims.
Nonmagnetic streptavidin mutein–immobilized CD4 Fab monomer via reversible binding site Z
A method in which the stationary phase chromatographic matrix is nonmagnetic or non-magnetizable and comprising a streptavidin mutein affinity reagent immobilized thereon; the receptor binding reagent comprises a Fab monomer fragment that binds CD4 and further comprises a binding partner C comprising a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment, capable of reversibly binding a binding site Z of the immobilized streptavidin mutein, wherein the binding site Z has a lower KD for biotin or a biotin analog than for the binding partner C.
Selection affinity chromatography complex formation with multimers of CD4 Fab fusion
Loading a sample comprising one or more CD4+ lymphocyte target cells onto a selection affinity chromatography column formed by loading the chromatographic matrix comprising the immobilized receptor binding reagent, wherein the complex comprises multimers of the monomeric Fab fragment fusion bound to the target cells, thereby reversibly immobilizing the one or more CD4+ lymphocyte target cells on the chromatographic matrix.
Competition reagent elution using biotin or biotin analog to release CD4 lymphocytes
Washing the selection affinity chromatography column to remove unbound cells, and eluting the bound CD4+ lymphocyte target cells by contacting the selection affinity chromatography column with a competition reagent selected from biotin or a biotin analog capable to disrupt the binding between the streptavidin-binding peptide of the receptor binding reagent and the streptavidin mutein, thereby displacing the receptor binding reagent and releasing the one or more CD4+ lymphocyte target cells in an eluate comprising the CD4+ lymphocytes, the Fab monomers, and biotin or a biotin analog.
Across the independent claim, the key inventive elements are a nonmagnetic stationary phase carrying a streptavidin mutein affinity reagent immobilized via a reversible binding site Z with a CD4-binding Fab monomer fusion, formation of a selection affinity chromatography complex with target-cell-associated multimers of the Fab fusion, and release by biotin or biotin analog competition elution yielding an eluate containing the target cells, Fab monomers, and biotin or biotin analog.
Stated Advantages
Enables release of target cells by disrupting reversible binding using a competition reagent.
Avoids covalent cleavages, thereby reducing need for removal steps.
Reduces removal needs including reducing reliance on magnetic beads.
Documented Applications
Isolation of CD4+ lymphocyte target cells using selection affinity chromatography with subsequent washing and competition reagent elution.
Isolation of CD8+ and CD4+ cells from human PBMC/whole blood and murine splenocytes using reversible enrichment concepts involving streptavidin/streptavidin mutein and related peptide/biotin competition release.
Use of a size-exclusion removal chromatography step to retain Fab/biotin components while isolating target cells.
Implementation in an apparatus format using serial selection/removal cartridges and chromatographic formats including a planar nitrocellulose matrix and a pipette tip format.
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