Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry.

Eluforsen (previously known as QR-010) is a 33-mer 2'-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3' exonuclease were observed in mouse liver in an in vitro incubation system and by either 3' exonuclease or 5' exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2'-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides.

Alkylamines are commonly used to improve both chromatographic and mass spectral performance of electrospray ionization liquid chromatography mass spectrometry based methods for the analysis of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatographic properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resolution and general chromatographic performance of fifteen different alkylamines for the separation of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatographic performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concentrations of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatography and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility experiments detecting three micron aggregates in the mobile phase at concentrations that are routinely used for LC-MS analysis of oligonucleotides. These aggregates are not detected at lower alkylamine concentrations where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.