Chromatographic isolation of cells and other complex biological materials
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Publication Number
US-12135265-B2
Publication Date
2024-11-05
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 describes traceless cell affinity chromatography (CATCH) for isolating target cells or other biological entities using a receptor binding reagent and an affinity chromatography stationary phase. Target cells are immobilized through reversible non-covalent binding between a binding partner C on the receptor binding reagent and binding site Z on an affinity reagent carried by the chromatography stationary phase, enabling capture without covalent attachment and without magnetic beads.
After capture, elution is achieved by competitive disruption of the reversible binding between binding partner C and binding site Z. This competitive disruption allows removal of the receptor binding reagent from the isolated cells and uses competition reagents to avoid retention of the binding components, while supporting gentle, viable isolation and subsequent purification steps using cartridge-based selection and removal arrangements.
The disclosed embodiments include affinity and/or gel filtration formats and selection/removal cartridge configurations for serial multi-step purification. Specific examples are described for CD4+ and CD8+ cell enrichment and the ability to remove Fab and biotin essentially completely using a second removal cartridge, with column formats as well as planar and pipette-based configurations for affinity and/or size-based separations.
Claims Coverage
The provided independent claim coverage includes one independent claim. It covers a CD4+ lymphocyte separation chromatography column with a stationary matrix architecture using an immobilized streptavidin mutein and a receptor binding reagent immobilized through a binding partner C to a binding site Z.
Traceless CD4+ affinity stationary matrix with immobilized streptavidin mutein
An affinity chromatography stationary matrix suitable for a CD4+ lymphocyte cell separation comprising immobilized thereon both an affinity reagent comprising a streptavidin mutein and a receptor binding reagent.
Fab monomer CD4 receptor binding reagent linked via streptavidin-binding peptide binding partner C
A receptor binding reagent comprising a Fab monomer fragment that binds CD4 on the surface of lymphocytes and a binding partner C comprising a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment, thereby enabling binding to the immobilized streptavidin mutein.
Reversible immobilization on binding site Z with differential binding to biotin/biotin analog
The binding partner C is bound to a binding site Z of the immobilized streptavidin mutein to immobilize the receptor binding reagent on the chromatography matrix, with the binding site Z having a lower dissociation constant for biotin or a biotin analog than for the binding partner C.
The claim coverage centers on a CD4+ lymphocyte affinity chromatography column where a receptor-binding Fab is immobilized via a streptavidin-binding peptide to an immobilized streptavidin mutein, with binding-site affinity designed to favor biotin or biotin-analog interaction for competitive disruption.
Stated Advantages
Gentle, viable isolation without magnetic beads.
Enables removal of the receptor binding reagent after elution by competitive disruption.
Supports serial multi-step purification using selection/removal cartridge arrangements.
Reports essentially complete removal of Fab and biotin using a second removal cartridge.
Documented Applications
CD4+ lymphocyte enrichment using an affinity chromatography stationary matrix configured as a chromatography column.
CD8+ enrichment using traceless cell affinity chromatography (CATCH) with reported purity and yield.
Use of selection/removal cartridge arrangements for serial multi-step purification, including a second removal cartridge for Fab and biotin removal.
Planar chromatography on nitrocellulose and pipette-tip chromatography in up-and-down mode for affinity and/or size-based separation.
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