Chromatographic separation of phosphorothioate oligonucleotides
AssigneeLynx Therapeutics, Inc.
InventorB. John Bergot
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Abstract
A method is provided for the preparation and/or analysis of synthetic phosphorothioate and -dithioate oligonucleotides. In particular, the method permits separation of fully sulfurized phosphorothioate or -dithioate oligonucleotides from incompletely sulfurized defect species on an anion exchange HPLC columns using concentration gradients of novel "soft base" anionic eluents, such as bromide, thiocyanate, and the like.
Claims15
A method of separating completely sulfurized phosphorothioate or phosphorodithioate oligonucleotides from a mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the method comprising the steps of:
- (a) impregnating a anion exchanger with the mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides;
- (b) passing through the anion exchanger a buffer solution comprising a concentration of a soft-base counter ion which monotonically increases with time to a sample-desorbing concentration;
- and
- (c) recovering the eluate from step (b).
A method of measuring the fraction of completely sulfurized phosphorothioate or phosphorodithioate oligonucleotides in a mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the method comprising the steps of:
- (a) impregnating a anion exchanger with the mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides;
- (b) passing through the exchanger a buffer solution comprising a concentration of a soft-base counter ion which monotonically increases with time to a sample-desorbing concentration so that a chromatogram of the eluted completely sulfurized and incompletely sulfurized phosphorothioate and phosphorodithioate oligonucleotides is formed;
- and
- (c) computing the ratio of the peak area corresponding to the completely sulfurized phosphorothioate or phosphorodithioate oligonucleotide on the chromatogram to the total area under all peaks corresponding to both completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides on the chromatogram.
Dependencies are inferred from the claim text.