Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Gilár, Martin; Fountain, Kenneth J.; Budman, Yeva; Holyoke, Jeffrey L.; Davoudi, Hamid; Gebler, John C.

doi:10.1089/154545703322460612

Cited by 99 publications

(81 citation statements)

References 43 publications

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“…Capillary gel electrophoresis (CGE) [7], strong anionexchange (SAX) chromatography [8], 31 P NMR spectroscopy [8,9], matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) [10,11], liquid chromatography coupled to electrospray ionization (ESI) mass spectrometry [12,13], and tandem mass spectrometry (MS/MS) [13][14][15] are among the analytical methods that have been used to determine the quality of oligonucleotides and characterize their impurities. Ion pair-reversed phase (IP-RP) HPLC coupled with a UV detector and electrospray ionization has been identified as one of the most suitable methods because of its ability to provide increased chromatographic resolution and reliable qualitative and quantitative information [6].…”

Section: Introductionmentioning

confidence: 99%

“…Ion pair-reversed phase (IP-RP) HPLC coupled with a UV detector and electrospray ionization has been identified as one of the most suitable methods because of its ability to provide increased chromatographic resolution and reliable qualitative and quantitative information [6]. Advanced molecular characterization is possible using high-resolution MS [16,17] and/or MS/MS instrumentation [13,18,19].…”

Section: Introductionmentioning

confidence: 99%

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A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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Abstract.A new method is presented for determining relationships between components in complex analytical systems. The method uses the mass differences between peaks in high resolution electrospray ionization (ESI) mass spectra. It relates peaks that share common mass differences. The method is based on the fundamental assumption that peaks in the spectra having the same exact mass difference are related by the same chemical moiety/substructure. Moreover, the presence (or absence/loss) of the same chemical moiety from a series of molecules may reflect similarities in the mechanisms of formation of each molecule. The determined mass differences in the spectra are used to automatically differentiate the types of components in the samples. Contour plots and summary plots of the summed total ion signal as a function of the mass difference are generated, which form powerful tools for the rapid and automated determination of the components in the samples and for comparisons with other samples. For the first time, in this work a unique profile contour plot has been developed that permits the interactive interrogation of the mass range by mass difference data matrix to obtain valuable information about components that share a common mechanism of formation, and all possible mechanisms of formation linked to a selected precursor molecule. The method can be used as an additional and complementary method to the existing analytical methods to determine relationships between components in complex chemical systems.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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“…There are many theoretical studies concerning the interaction of oligonucleotides within the surface of stationary phase [8][9][10]. Most of them include complex mechanisms that comprise different interactions in hydro-organic solvents [1][2][3][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…Utilization of this packing material does not achieve high efficiency and selectivity in the case of the studied compounds [8][9][10]. Thus, another type of stationary phase was chosen for the present study, namely alkylamide.…”

Section: Introductionmentioning

confidence: 99%

Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase

2015

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Abstract:The main aim of the present investigation was to determine retention behavior and interactions of oligonucleotides with alkylamide stationary phase. Five oligonucleotides, differing in the sequence, were tested. Changes in the composition of the mobile phase, i.e. pH (5.0−7.0) and buffer concentration (30−75 mM) were investigated in detail. In addition, the hydrophobic and electrostatic parameters were measured for the different pH's and salt concentrations. The theoretical model of interactions between individual elements of the separation system, (e.g. solute, stationary phase, and mobile phase) based on zeta potential measurements, hydrophobic and electrostatic parameters calculation, and molecular modeling, has been considered.

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“…Nevertheless, contamination of the target sequence with truncated sequences, partially deprotected sequences as well as partially desulfurized species can be observed (28,(33)(34)(35). Therefore, prior to use, we decided to check the quality of the purchased oligonucleotide with MS.…”

Section: Resultsmentioning

confidence: 99%

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Erb

Leithner²,

Bernkop‐Schnürch³

et al. 2012

AAPS J

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Abstract. Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversedphase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra-and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.

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Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection

Cited by 99 publications

References 43 publications

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

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