Analysis of antisense oligonucleotides by on‐capillary isotachophoresis and capillary polymer sieving electrophoresis

Abstract

An attempt was made to evaluate the stability of an antisense oligonucleotide against nucleases present in HBL 100ras cells. To detect nanomolar concentrations of the oligonucleotide, a sensitive detection system was required. A combination of capillary electrophoresis/laser-induced fluorescence (CE-LIF) with fluorescence derivatization did not improve the sensitivity significantly and also resulted in loss of separation of the derivatized sample. On-column isotachophoresis for the preconcentration of oligonucleotide samples in DB-17 coated capillaries filled with hydroxyethyl cellulose solution could be an alternative. The isotachophoresis (ITP) step allows injection of up to 40% of the capillary volume without loss in peak resolution and peak efficiency. Using ITP-capillary polymer sieving electrophoresis (CPSE), the limit of quantitation at a signal-to-noise ratio of 10 was 73 ng/mL for a 12-mer oligonucleotide. Using these conditions, the gain in sensitivity was 125.

On-column electroextraction and separation of antisense oligonucleotides in human plasma by capillary gel electrophoresis

“…Similar to CE in general, however, the concentration has to be fairly high, in the nanomolar range, corresponding to low microgram-high nanogram of oligo per milliliter sample. To enhance the detection sensitivity of antisense in CGE laser-induced fluorescence detection [13] and isotachophoresis in partially polymer-filled capillaries [36,37] have been reported.…”

Recent progress in capillary isotachophoresis

“…For anions, two different systems were reported (LE = BGE: 10 mM HCl adjusted to pH 7.4 with ammonia; TE: 100 mM glycine adjusted to pH 8.6 with ammonia) [90], (LE: 0.1±5% ammonium hydroxide; TE = BGE: 2 mM ammonium acetate, 1% acetic acid, pH 2.9) [91]. Further, one can mention here peptides in plasma (LE = BGE: 10 mM ammonium acetate adjusted to pH 5.0 with acetic acid; TE: 10 mM acetic acid, both 50% v/v methanol) [92] and antisense oligonucleotides (LE: 50 mM HCl; TE: 50 mM butyric acid; BGE: 50 mM HCl, 6 M urea, 5% HEC, all adjusted to pH 7.5 with Tris) [93]. A study of the effect of electromigration dispersion in CZE with transient ITP of several basic model proteins and interleukin-6 (rhIL-6) has led to the introduction of an electromigration dispersion factor that measures the level of peak asymmetry, increasing with the analyte amount and mobility [94].…”

Recent progress in capillary isotachophoresis

“…For anions, two different systems were reported (LE = BGE: 10 mM HCl adjusted to pH 7.4 with ammonia; TE: 100 mM glycine adjusted to pH 8.6 with ammonia) [90], (LE: 0.1±5% ammonium hydroxide; TE = BGE: 2 mM ammonium acetate, 1% acetic acid, pH 2.9) [91]. Further, one can mention here peptides in plasma (LE = BGE: 10 mM ammonium acetate adjusted to pH 5.0 with acetic acid; TE: 10 mM acetic acid, both 50% v/v methanol) [92] and antisense oligonucleotides (LE: 50 mM HCl; TE: 50 mM butyric acid; BGE: 50 mM HCl, 6 M urea, 5% HEC, all adjusted to pH 7.5 with Tris) [93]. A study of the effect of electromigration dispersion in CZE with transient ITP of several basic model proteins and interleukin-6 (rhIL-6) has led to the introduction of an electromigration dispersion factor that measures the level of peak asymmetry, increasing with the analyte amount and mobility [94].…”