Polynucleotide purification with monolith columns
AssigneeAlexion Pharmaceuticals, Inc.
InventorsJared DAVIS, Joanna DEBEAR, Christopher CHENG
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Abstract
Described herein are methods of purifying polynucleotides, e.g., imRNA and oligonucleotides, e.g., probes, primers and siRNA, using monolithic columns with immobilized ligands coupled to the monolithic column. Also described are monolithic columns for purifying polynucleotides from a sample; and methods of preparing such columns.
Claims32
A method of purifying a polynucleotide from a sample, the method comprising:
- a) loading the sample onto a monolithic matrix comprising a ligand comprising:
- i) a reactive moiety coupled to the monolithic matrix, and
- ii) an affinity moiety having the formula —C12-dT18, wherein dT18 is a poly-A binding oligomer consisting of 18 deoxythymidine monomers that binds to the polynucleotide, —C12— is a 12-carbon linker chain positioned between the poly-A binding oligomer and the reactive moiety, wherein the ligand is immobilized to the monolithic matrix via the reactive moiety;
- b) allowing for the polynucleotide to bind to the ligand;
- and
- c) eluting the polynucleotide from the monolithic matrix after one or more contaminants have been substantially separated from the bound polynucleotide.
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A method of separating a formulated polynucleotide from free polynucleotide, the method comprising:
- a) loading a sample onto a monolithic matrix comprising a ligand comprising:
- i) a reactive moiety coupled to the monolithic matrix, and
- ii) an affinity moiety having the formula —C12-dT18, wherein dT18 is a poly-A binding oligomer consisting of 18 deoxythymidine monomers that binds to the free polynucleotide but not the formulated polynucleotide, —C12— is a 12-carbon linker chain positioned between the poly-A binding oligomer and the reactive moiety, wherein the ligand is immobilized to the monolithic matrix via the reactive moiety;
- and
- b) collecting the formulated polynucleotide from the column while the free polynucleotide remains immobilized on the monolithic matrix.
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A preparative-scale column for purifying a polynucleotide from a sample, said column comprising:
- c) a monolithic matrix;
- and
- d) a ligand coupled to the monolithic matrix, wherein the ligand comprises a reactive moiety and an affinity moiety having the formula —C12-dT18, wherein —C12— is a 12-carbon linker chain positioned between the reactive moiety and dT18, wherein the dT18 is a poly-A binding oligomer consisting of 18 deoxythymidine monomers, and wherein the affinity moiety is coupled to the monolithic matrix at the carbon linker via the reactive moiety;
- wherein the column has a column volume of at least 0.34 mL and is configured to allow a flow rate of at least 1 mL/min.
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Dependencies are inferred from the claim text.