The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

Abstract

A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C(22)) to ODN at 5'-termini (denoted as 5'C(22)-Flu) or 3'-termini (denoted as 3'C(22)-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5'C(22)-Flu. As expected, the method developed was successfully applied to the analysis of crude 3'C(22)-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

“…IP‐RP‐HPLC has been conducted using several alkylammonium salts as ion pairing agents, including tripentylammonium acetate (Capaldi et al, ; Gaus et al, ), tributylamine (Roussis, ), triethylammonium formate (Gaus et al, ), triethylammonium acetate (Fearon et al, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Rentel et al, ), tetrabutylammonium phosphate (Metelev & Agrawal, ), tributylammonium acetate (Kurata et al, ; Rentel et al, ) and hexylammonium acetate (Cramer et al, ; Smith & Beck, ). Moreover using a buffering system containing the ion pairing agents and hexafluoroisopropanol and triethylamine (Farand & Beverly, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Li et al, ; Liu et al, ; Nikcevic et al, ) and diisopropylethylamine and hexafluoroisopropanol (Chen & Bartlett, ; McGinnis et al, ) has provided significant chromatographic separation and higher signal intensity, as well as higher selectivity and chromatographic resolution (Apffel et al, ; Basiri & Bartlett, ; McGinnis et al, ). Nonion‐pairing conditions have also been applied using acetonitrile with formic acid or ammonium acetate (Cohen et al, ; Culf et al, ) or NH 4 HCO 3 , EDTA (Gilar et al, ) or ammonium formate (An et al, ; Suzuki et al, ).…”

“…A UHPLC–MS/MS assay has been adopted for the simultaneous quantitation of a full‐length oligonucleotide and its ( n − 1)‐mer (Chen & Bartlett, ). Additionally, MALDI‐TOF‐MS has also been utilized for the characterization and identification of oligonucleotide impurities (Ball & Packman, ; Li et al, ; Liu et al, ).…”

“…Similar separations have been accomplished using IP‐RP‐HPLC, resulting in improved selectivity and reproducibility when compared with capillary gel electrophoresis and ion‐exchange HPLC for the same set of compounds (Li et al, ). The PO analogs have been identified using both ESI‐MS (Gilar et al, ; Roussis, ; Smith & Beck, ) and MALDI‐TOF‐MS (Li et al, ; Liu et al, ; Yang et al, ). In addition, 31 P–NMR spectroscopy has been used for further examination of PO analogs (Bergot & Egan, ; Smith & Beck, ; Srivatsa et al, ; Yang et al, ).…”

Delivery and Biological Activity of Therapeutic miRNAs and miRNA Modifiers

“…Figure 39.4 outlines some recently proposed DNA analog employed in therapeutic interventions, such as 2′-deoxyoligonucleotides [54], 2′-O-methyl-modified oligoribonucleotides (2′-OMes) [55], cholesterol moiety-conjugated 2′-OMe [56,57], locked nucleic acid (LNA-) -modified oligonucleotides [58], oligonucleotides containing 2′-O-methoxyethyl (2′-MOE) [59], 2′-flouro (2′-F) [60], and phosphorothioate backbone modifications [61,62]. Novel molecules were developed that, when compared with standard oligonucleotides, exhibit improved characteristics in respect to hybridization efficiency, stability in biological fluids, and suitability for delivery to target cells or tissues.…”

The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

“…Thus, quality control of oligonucleotides becomes vital in their synthesis. One of the major assay technologies for the analysis of oligonucleotides is chromatography, including ion‐exchange chromatography, size‐exclusion chromatography, reversed‐phase high‐performance liquid chromatography (HPLC), affinity chromatography, ion‐pairing reversed‐phase (IP‐RP) HPLC, and hydrophilic interaction liquid chromatography (HILIC) …”