Hydrophobic interaction chromatography for purification of oligonucleotides
AssigneeBiogen MA Inc.
InventorsRobert S. Gronke, Ratnesh S. Joshi
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Abstract
The invention herein describes a method to purify a target oligonucleotide using hydrophobic interaction chromatography (HIC). The method includes adding a salt to a mixture of the target oligonucleotide and product-related impurities, applying the diluted mixture, at a particular dynamic loading capacity, to the hydrophobic interaction chromatography resin (or hydrophobic adsorbent), washing the hydrophobic adsorbent with an aqueous salt solution, eluting the target oligonucleotide with a eluting solution, and collecting the eluent comprising the target oligonucleotide.
Claims18
A method for separating a target oligonucleotide from a mixture containing the target oligonucleotide and a product-related impurity, the method comprising the steps of:
- a) adding a salt to the mixture, wherein the salt is ammonium sulfate;
- b) contacting the diluted mixture with a hydrophobic adsorbent at a dynamic loading capacity of about 32 to about 78% of the capacity of the hydrophobic adsorbent wherein the hydrophobic adsorbent comprises a phenyl or a hexyl crosslinked agarose;
- c) washing the hydrophobic adsorbent with an aqueous salt solution;
- d) eluting the target oligonucleotide with an eluting solution;
- and
- e) collecting the eluent comprising the target oligonucleotide;
- wherein the product-related impurity comprises at least one N−1 impurity, thereby separating the target oligonucleotide from the product-related impurity.
A method for separating a target oligonucleotide from a mixture containing the target thiolated oligonucleotide and a product-related impurity, the method comprising the steps of:
- a) adding a salt to the mixture, wherein the salt is ammonium sulfate;
- b) contacting the diluted mixture with a hydrophobic adsorbent at a dynamic loading capacity of about 40 to about 100% of the capacity of the hydrophobic adsorbent, wherein the hydrophobic adsorbent comprises a phenyl or a hexyl crosslinked agarose;
- c) washing the hydrophobic adsorbent with an aqueous salt solution;
- d) eluting the target oligonucleotide with an eluting solution;
- and
- e) collecting the eluent comprising the target oligonucleotide;
- wherein the product-related impurity comprises at least one P═O impurity, thereby separating the target oligonucleotide from the product-related impurity.
Dependencies are inferred from the claim text.