Bioanalytical LC–MS of Therapeutic Oligonucleotides

Dongen, W. D. van; Niessen, W.M.A.

doi:10.4155/bio.11.8

Cited by 64 publications

(25 citation statements)

References 87 publications

(238 reference statements)

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“…Various approaches for ODN extraction from the biological matrix have been used including protein precipitation, liquid-liquid extraction (LLE) and SPE, though each method has its own shortcomings and compound-dependent success rates [27,[29][30][31][32][33].…”

mentioning

confidence: 99%

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

et al. 2017

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Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach. In recent years, oligonucleotide drugs (ODNs) have gained popularity owing to their ability to target specific genes and provide specificity. ODNs comprise an oligonucleotide sequence, containing 10-50 nucleotides [1,2]. Numerous ODNs have been investigated so far [1][2][3][4]. But only six ODNs (fomivirsen, pegaptanib, mipomersen, eteplirsen, nusinersen and defibrotide sodium) have been approved by the US FDA since 1998 [5].Pharmaceutical development of ODNs has several technical challenges such as maintaining in vivo stability, ensuring delivery and bioavailability, and minimizing off-target effects. These challenges can be minimized by chemical modifications of ODNs to enhance their pharmacokinetic and pharmacodynamic properties [4,6]. These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].Imetelstat is a novel, first-in-class telomerase inhibitor ODN [12], being investigated for the treatment of hematologic myeloid malignancies [13,14] and certain solid tumors [12,15,16]. Imetelstat is a 13-mer oligonucleotide N3 -P5 thio-phosphoramidate (NPS) with a covalently linked C16 (palmitoyl) lipid moiety at the 5 -end (Figure 1; Supplementary Table 1). Addition of a lipid chain and the modified oligonucleotide backbone enables imetelstat to penetrate cells and tissues, with high biodistribution into normal and malignant cells [12].Imetelstat differs from antisense oligonucleotides in its mechanism of action. Imetelstat is complementary to the template region of the RNA component of telomerase, to which it binds with high affinity and specificity, and directly competes with telomere binding, thereby inhibiting telomerase activity [17]. Thus, imetelstat acts as a classical ac...

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confidence: 99%

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

et al. 2017

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“…Nowadays, MALDI-MS and ESI-MS are most frequently applied in the analysis of oligonucleotides [264,265]. In some applications, e.g., impurity profiling of synthetic oligonucleotides and the (bio) analysis of therapeutic (antisense) oligonucleotides [266], ESI-MS seems to be preferred, as it can be applied in combination with LC-MS [267].…”

Section: Oligonucleotidesmentioning

confidence: 99%

“…The negative-ion mode is indispensable for the MS analysis of oligonucleotides [264][265][266][267][268][269]. In ESI-MS, larger oligonucleotides will show multiple-charge ions, with typically one charge per 0.7-1 kDa.…”

Section: Oligonucleotidesmentioning

confidence: 99%

Ion/Molecule Attachment Reactions: Mass Spectrometry

Fujii¹

2015

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“…The obtained ER values are below the minimum value of 50 % recommended for quantification of small bioorganic molecules (49). With respect to SPE of oligonucleotides, ER seem to be quite excellent (13). ME values were determined by comparing the signal response of post-extraction spiked samples and non-matrixprepared samples.…”

Section: Methods Developmentmentioning

confidence: 99%

“…Commonly applied techniques for qualitative and quantitative analysis of therapeutic oligonucleotides include immunoassays, quantitative polymerase chain reaction, electrophoresis, liquid chromatography (LC), mass spectrometry (MS), and combinations thereof (4,(8)(9)(10)(11)(12)(13). Particularly immunoassays provide very low detection limits.…”

Section: Introductionmentioning

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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Bioanalytical LC–MS of Therapeutic Oligonucleotides

Cited by 64 publications

References 87 publications

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

Ion/Molecule Attachment Reactions: Mass Spectrometry

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

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