Chromatographic isolation of cells and other complex biological materials
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Publication Number
US-10228312-B2
Publication Date
2019-03-12
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 relates to a traceless cell affinity chromatography approach for isolating a target cell having a receptor molecule on the surface of the target cell. The method uses chromatography on a stationary phase that includes a gel filtration matrix and/or an affinity chromatography matrix. The sample comprises one or more target cells, a competition reagent, and a receptor binding reagent having a binding site B and a binding partner C.
Binding site B specifically binds to the receptor molecule and binding partner C reversibly binds to an affinity reagent through a non-covalent reversible complex. The stationary phase comprises an affinity reagent that includes streptavidin, a streptavidin mutein, avidin, an avidin mutein, or a mixture thereof, together with a plurality of binding site Z. Binding sites Z specifically bind to the binding partner C and to the competition reagent, enabling incubation under conditions sufficient to allow binding of binding site Z with binding partner C and with the competition reagent.
In related embodiments, the invention uses first and second stationary phases to form and disrupt reversible immobilization of target cells. A first stationary phase immobilizes an affinity reagent having a plurality of first binding site Z that forms a non-covalent reversible complex with the binding partner C, thereby reversibly immobilizing the target cells, and a competition reagent is loaded to disrupt the non-covalent reversible complex. The elution sample is then exposed to chromatography on a second stationary phase having a gel filtration matrix and/or an affinity chromatography matrix, with a second affinity reagent having a plurality of second binding site Z that bind the binding partner C, isolating at least one target cell that is free from a bound receptor binding reagent.
Claims Coverage
The independent claims cover three main inventive approaches: a single-step isolating method with gel filtration and/or affinity chromatography and competition-driven release, a two-stage chromatographic isolation method with competition disruption and subsequent recovery free of bound receptor binding reagent, and a binding/immobilization method using a monovalent receptor binding reagent and an affinity reagent bearing immobilized binding sites Z.
Traceless isolation via gel filtration and/or affinity chromatography with competition disruption
A method of isolating a target cell by exposing a sample comprising one or more target cells, a competition reagent, and a receptor binding reagent to chromatography on a stationary phase, where the stationary phase comprises a gel filtration matrix and/or an affinity chromatography matrix including an affinity reagent with binding sites Z that bind binding partner C and the competition reagent; incubating to allow binding of binding site Z with binding partner C and with the competition reagent; and collecting an eluate comprising at least one target cell free from a bound receptor binding reagent.
Two-stage chromatographic isolating with first disruption and second affinity recovery
A method of chromatographically isolating a target cell by providing a sample with a receptor binding reagent having binding site B and binding partner C, exposing the sample to chromatography on a first stationary phase with an immobilized first affinity reagent having first binding site Z forming a non-covalent reversible complex with binding partner C to reversibly immobilize the target cells, loading a competition reagent that disrupts the complex via specific binding to the first binding site Z, recovering an elution sample, and exposing the elution sample to chromatography on a second stationary phase comprising a gel filtration matrix and/or an affinity chromatography matrix having a second affinity reagent with second binding site Z binding the binding partner C to isolate at least one target cell free from a bound receptor binding reagent.
Reversible immobilization using a monovalent receptor binding reagent and immobilized binding site Z
A method of isolating a target cell by contacting a sample comprising one or more target cells with a receptor binding reagent having a monovalent binding site B and a binding partner C, wherein the receptor binding reagent is selected from a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like functions, an aptamer, or an MHC molecule; exposing the sample to chromatography on a stationary phase having the affinity reagent comprising the binding site Z immobilized thereon, where binding site Z forms a non-covalent reversible complex with binding partner C and binding site B binds the receptor molecule on the target cell surface, thereby reversibly immobilizing the one or more target cells on the stationary phase.
Across the independent claims, the core coverage is directed to reversible immobilization of target cells using binding partner C with immobilized binding sites Z on an affinity reagent, and to disruption/recovery steps that yield target cells free from bound receptor binding reagent, including a multi-stage approach using first and second stationary phases.
Stated Advantages
The collected eluate comprises target cells that are free from a bound receptor binding reagent.
Documented Applications
Enriching and purifying CD8+ T-cells and CD4+ T-cells, including use with PBMC and whole blood contexts described in the document summary.
Serial purification cycles using apparatus concepts including selection/removal cartridge arrangements and controlled operation.
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