Target Cell Isolation via Reversible Multivalent Binding Complex
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Solution Overview
Problem
Current cell isolation methods, such as chromatography and magnet-assisted cell sorting, face challenges including low yield, high cost, and the need for complex equipment, as well as potential compromise of cell viability and effector function due to residual reagents, particularly when isolating complex cell populations like regulatory T cells or central memory T-cells.
Innovation Solution
A method involving a receptor molecule binding reagent with a binding site capable of specific binding to a target cell surface receptor molecule, combined with a soluble multimerization reagent that forms a multivalent binding complex, allowing reversible immobilization on a solid phase via a ligand, enabling efficient and gentle isolation of target cells without residual reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional chromatography sorbents with large pores are used to isolate cells, then cell accessibility is improved, but the surface area available for ligand immobilization is reduced due to diffusional limitations
Solution Approach 1:
The patent employs porous glass beads with controlled pore sizes and surface treatments to achieve optimal balance between cell accessibility and ligand immobilization surface area. The porous structure allows cells to penetrate and access binding sites while providing sufficient surface area for receptor ligand attachment.
Solution Approach 2:
The invention uses composite sorbent materials combining porous glass beads with immobilized receptor ligands, creating a multifunctional matrix that simultaneously provides structural support, cell accessibility, and specific binding capacity for cell isolation.
2Productivity
If a large excess of receptor binding ligands is used to compensate for pore inaccessibility, then cell binding capacity is improved, but the cost and complexity of reagent production increases
Solution Approach 1:
The patent optimizes ligand density and pore size parameters to achieve efficient cell binding with minimal ligand excess. By carefully controlling the concentration and distribution of immobilized receptor ligands on the sorbent surface, the method maximizes binding capacity while minimizing reagent requirements.
3Reliability
If monolithic sorbents are used for affinity chromatography of cells, then cell viability is improved, but additional procedures are required to remove elution compounds from cells
Solution Approach 1:
The patent extracts and removes the multimerization reagent and competing compound from the cell isolation process through sequential washing steps, leaving only the specifically bound target cells on the sorbent. This eliminates the need for harsh elution conditions that could compromise cell viability while still enabling effective cell separation.
4Speed
If flow cytometry with high pressure cell sorting is used, then cell isolation speed is improved, but cell effector function is compromised
Solution Approach 1:
The patent replaces the mechanical high-pressure sorting system of flow cytometry with a chemical affinity-based separation method using immobilized receptor ligands. Cells are isolated through specific binding interactions at low pressure, maintaining their functional integrity while achieving rapid separation through optimized sorbent design.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method provides a rapid, efficient, and standardized protocol for isolating target cells, improving yield and purity while maintaining cell viability and allowing for serial positive selection, independent of the binding characteristics of the receptor molecule and its reagent, and can be automated for GMP conditions.
Implementation Method 1
a receptor molecule binding reagent, the receptor molecule binding reagent comprising a binding site B and a binding partner C, wherein the binding site B comprised in the receptor molecule binding reagent is capable of specifically binding to the receptor molecule on the target cell surface
Implementation Method 2
a (soluble) multimerization reagent, wherein the multimerization reagent comprises two or more binding sites Z capable of reversibly binding to the binding partner C comprised in the receptor molecule binding reagent
Implementation Method 3
contacting the multivalent binding complex of target cell, receptor molecule binding reagent and multimerization reagent with a solid phase, the solid phase comprising the ligand L, thereby allowing reversible immobilization of the target cell on the solid phase via the binding between the ligand L and the ligand binding partner LB
Data Source
AI summary
The invention provides new methods of isolating target cells using a solid phase, the solid phase comprising a ligand L wherein the ligand L is capable of specifically binding a ligand binding partner LB, the ligand binding partner LB being present in a receptor molecule binding reagent or a multimerization reagent used for isolating target cells. The invention also provides corresponding new arrangements and devices for isolating a target cell from a sample.