Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides

Journal of Chromatography. a
Stilianos G RoussisClaus Rentel

Abstract

In an effort to improve separation of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tributyl and hexyl amine provided the best chromatography, as small amines tended to broaden peaks due to the separation of diastereoisomers. For phosphate diester oligonucleotides, the best separations were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full separation of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addition of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak separation.

Citations

Feb 1, 2020·Journal of Pharmaceutical and Biomedical Analysis·Alexandre GoyonKelly Zhang
Mar 12, 2021·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Stilianos G RoussisClaus Rentel
Jun 9, 2021·Journal of Chromatography. a·Martin EnmarkTorgny Fornstedt
Aug 20, 2020·Journal of the American Society for Mass Spectrometry·J Michael SuttonMichael G Bartlett

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