Use of imac to improve rna purity
AssigneeModernaTX, Inc.
InventorMatthew Burak
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Abstract
Described are methods and apparatuses for isolating and purifying mRNA using Immobilized Metal Affinity Chromatography (IMAC). The methods and apparatuses use IMAC matrices and IMAC conditions designed to selectively bind and elute mRNA, and are useful, for example, for isolating and purifying mRNA from in vitro-transcribed compositions. The methods and apparatuses also are useful for isolating and purifying linear or circular RNA having a poly(A) tail.
Claims30
A method of purifying a polynucleotide having a poly(A) sequence, comprising:
- contacting a composition comprising a polynucleotide having a poly(A) sequence with an immobilized metal affinity chromatography (IMAC) ligand, wherein the IMAC ligand binds the polynucleotide having a poly(A) sequence to form an IMAC-polynucleotide complex;
- separating the composition from the IMAC-polynucleotide complex;
- and
- recovering purified polynucleotide from the IMAC-polynucleotide complex,
- wherein the polynucleotide comprises a poly(A) sequence having a length of about 20 adenine nucleotides or longer.
The method of any one of the preceding claims, wherein the polynucleotide is an mRNA.
The method of any one of the preceding claims, wherein the IMAC ligand comprises an amine-carboxylic acid chelating group and a metal ion.
The method of any one of the preceding claims, wherein the IMAC ligand comprises a metal ion selected from Cu2+, Zn2+, Ni2+, and Co2+.
The method of any one of the preceding claims, wherein the IMAC ligand is immobilized on a substrate.
The method of any one of the preceding claims, wherein the contacting comprises passing the composition through a chromatography column containing the IMAC ligand.
The method of any one of the preceding claims, wherein the composition contacted with the IMAC ligand has a pH<7 and >5.
The method of any one of the preceding claims, wherein the composition contacted with the IMAC ligand has a pH of about 6.
The method of any one of the preceding claims, wherein the composition contacted with the IMAC ligand comprises NaCl at a concentration of from about 100 mM to about 250 mM.
The method of any one of the preceding claims, wherein the composition contacted with the IMAC ligand comprises ethanol at a concentration of about 10% v/v.
The method of any one of the preceding claims, wherein recovering purified polynucleotide from the IMAC-polynucleotide complex is effected using an elution buffer.
The method of claim 19, wherein the elution buffer comprises imidazole, has a pH of about 8, and comprises NaCl at a concentration of about 200 nM.
The method of any one of the preceding claims, wherein the polynucleotide has a length of from about 50 to about 5000 nucleotides.
The method of any one of the preceding claims, wherein the polynucleotide has a length of from about 3000 to about 5000 nucleotides.
The method of any one of the preceding claims, wherein the poly(A) sequence has a length of from about 50 to about 300 adenine nucleotides.
The method of any one of the preceding claims, wherein the composition comprises a histidine-tagged protein, and wherein the method is effective to purify the polynucleotide from the histidine-tagged protein.
The method of any one of the preceding claims, wherein the composition comprises tailless RNA, and wherein the method is effective to purify the polynucleotide from the tailless RNA.
The method of any one of the preceding claims, wherein the polynucleotide is an mRNA and the composition is an in vitro transcribed-(IVT) mRNA comprising the mRNA and histidine-tagged T7 RNA polymerase, wherein the method is effective to purify the mRNA from the T7 RNA polymerase.
The method of any one of the preceding claims, wherein the polynucleotide is an mRNA and the composition is an in vitro transcribed-(IVT) mRNA composition comprising the mRNA and linearized DNA plasmid, histidine-tagged T7 RNA polymerase, and nucleotide triphosphates.
The method of any one of the preceding claims, wherein the composition comprises a chelating agent, and the method further comprises, prior to contacting the composition with the IMAC ligand, conducting a buffer exchange to remove chelating agent from the composition.
The method of any one of the preceding claims, further comprising a heat denaturation step.
The method of any one of the preceding claims, wherein the composition does not contain DNA.
An immobilized metal affinity chromatography (IMAC) column comprising an IMAC-polynucleotide complex comprising an IMAC ligand bound to a polynucleotide having a poly(A) sequence, wherein the poly(A) sequence has a length of about 20 adenine nucleotides or longer.
Dependencies are inferred from the claim text.