Oligonucleotide Bioanalysis: Sensitivity Versus Specificity
2011; Future Science Ltd; Volume: 3; Issue: 12 Linguagem: Inglês
10.4155/bio.11.111
ISSN1757-6199
Autores Tópico(s)CRISPR and Genetic Engineering
ResumoBioanalysisVol. 3, No. 12 EditorialFree AccessOligonucleotide bioanalysis: sensitivity versus specificityLaixin WangLaixin WangTandem Labs, a LabCorp company, 1121 East 3900 South, Suite C-105, Salt Lake City, UT 84124, USA. Search for more papers by this authorEmail the corresponding author at Laixin.Wang@Labcorp.comPublished Online:16 Jun 2011https://doi.org/10.4155/bio.11.111AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: bioanalysishybridization-ELISALC–MS/MSoligonucleotidesiRNAUPLCThe bioanalysis of oligonucleotide therapeutics is constantly evolving, primarily driven by drug development needs and regulatory requirements. The technologies have been comprehensively reviewed by Tremblay and Oldfield [1]. Each method has been used for different study purposes for a period of time. To support preclinical and clinical studies, assay robustness/ruggedness, sensitivity and specificity are critical features for obtaining reliable data from precious samples in a timely manner. With recent advances in analytical technologies, currently the three major technologies supporting the bioanalysis of oligonucleotide therapeutics, especially in good laboratory practice (GLP) environments, are hybridization-ELISA, LC–MS/MS and UPLC–UV (photodiode array).Hybridization-ELISA: less specific but sensitive for large oligonucleotides, no metabolite informationGenerally speaking, hybridization-ELISA (also known as hybridization immunoassay) provides the best assay sensitivity and throughput compared with other methods. Hybridization assays usually involve the hybridization of the target oligonucleotide to a capture probe (immobilization) and/or to a detection probe (signaling). Therefore, the assay requires little or no sample clean-up. But the assay has a narrow calibration range (usually 20–50-fold) and its robustness relies heavily on the quality of the reagents. The sensitivity and specificity of the assay depend on the choice of the methods/reagents and the structures of the target oligonucleotides.For a ‘sandwich hybridization’ assay, the LLOQ can be as low as 100 pg/ml in plasma [2]. However, the target oligonucleotide needs to be long enough (usually >20-mer) to allow both the capture probe and the detection probe to bind stably to different parts of the molecule. This method is usually unable to distinguish the large metabolites (truncated n-1 and n-2 metabolites) from the full-length target oligonucleotide.The ‘hybridization-ligation’ assay is much more specific and has comparable sensitivity [3]. The length requirement of the target is also much shorter, as the target oligonucleotide binds to part of the capture probe (also known as the template probe). The capture probe has a generic 5´-end overhang, which is complementary to a 5´-phosphorylated ligation probe. As the enzyme ligation efficiency is very low in the presence of a gap, the 3´-end truncated metabolites are difficult to detect using the ligation-based assay. The 5´-end metabolites, however, will interfere with the detection of the full-length oligonucleotide.In theory, the ‘nuclease-based hybridization’ assay is more specific than the ‘hybridization-ligation’ assay. It uses the properties of a single-strand-specific nuclease (S1 nuclease) to degrade the free capture probe and the non-fully matched hybrids [101]. Therefore, only the full length target oligonucleotides are expected to be detected. However, in practice, the sequence context and the intrinsic characteristics of the S1 nuclease commonly used with the digestion assay may also allow the detection of n-1 truncated metabolites [4].The ‘competitive hybridization’ assay is an option if the sandwich or ligation assays are not feasible, due to the length or structure limit of the analyte oligonucleotide [5]. In addition to the low assay specificity, the reported limit of the quantification of a competitive hybridization assay is 900 pM (∼5 ng/ml) in plasma for a 15-mer phosphodiester oligonucleotide [6].LC–MS/MS: specific & sensitive for small oligonucleotidesLC–MS/MS has emerged as a powerful tool to quantify a variety of oligonucleotide therapeutics in biological matrices [6]. Contrary to hybridization-ELISA assays, LC–MS/MS is especially advantageous for quantifying relatively small oligonucleotide therapeutics (25-mer or shorter [7]. With three levels of discrimination (chromatography separation, molecular weight identification and product ion characterization) LC–MS/MS is one of the most specific bioanalytical methods for oligonucleotide therapeutics. Generally speaking, the sensitivity of an LC–MS/MS assay decreases when the size of an oligonucleotide increases due to the limit of LC resolution and the additional number of molecular charge states in the MS source [7]. Depending on the size of the target oligonucleotides, a 5–10 ng/ml LLOQ has been achieved from 100-µl plasma samples [8,9]. Compared with hybridization-ELISA assays, LC–MS/MS methods have a much wider dynamic range (up to three orders of magnitude) and no special reagents are required. It can also quantify intact double-stranded oligonucleotides, such as siRNA, by measuring either sense- or antisense-strand RNA. Many LC–MS/MS methods have been developed and validated to support preclinical and clinical studies of both single-stranded DNA oligonucleotides and double-stranded siRNAs [7].In addition to accurate and specific quantitations of the full-length oligonucleotides, the metabolites can also be analyzed simultaneously using the same LC–MS/MS assay if the standard reference materials are available [8]. In the meantime, the same LC–MS/MS platform can be used to analyze delivery vehicles, such as lipids or polymers in the study samples. Therefore, if sensitivity is adequate, LC–MS/MS is poised to become the first choice for the bioanalysis of oligonucleotide therapeutics.LC–MS/MS has become a standard technique for the determination of small-molecule drugs in biological fluids for more than a decade. However, until recently, it has been less successful for the quantitative bioanalysis of therapeutic oligonucleotides due to the many challenges associated with their unique physicochemical and biological properties [10]. These challenges include: ▪ Multiple charge states and cation adducts in the ion source;▪ Nonspecific binding to the components in biological matrices;▪ Adsorption to LC column and injector systems;▪ Chemical and enzymatic instability during sample storage and sample processing;▪ Nonspecific binding to container surfaces.Overcoming these challenges has been the key for the successful development and validation of an LC–MS/MS assay. As oligonucleotides are large polyanionic molecules they tend to bind matrix components, especially cationic components, which significantly affects the LC separation and MS ionization of the analyte oligonucleotides. Therefore, unlike the hybridization-ELISA assays, the biological samples need to be prepared and extracted appropriately prior to being analyzed via LC–MS/MS. Liquid–liquid extraction (LLE) with phenol/chloroform followed by SPE has been reported with recoveries greater than 70% for single-stranded DNA oligonucleotides [8]. A single step phenol/chloroform LLE extraction was found to be able to provide even better and more consistent recoveries from plasma and urine samples for all tested single-stranded oligonucleotides [9]. The key is to add the appropriate amount of ammonium hydroxide or other disrupting reagents to the sample to break down the oligonucleotide/matrix binding complexes prior to proceeding with phenol/chloroform extraction. In addition, it is important to keep some competitive component(s), such as EDTA, in the extracts to reduce the nonspecific binding of the oligonucleotide to the LC system and, therefore, improve the peak shapes and prolong the column lifetime. For the same reasons, the single-step LLE extracts can usually be stored longer (up to one week at 1–8°C) in plastic plates/vials than corresponding extracts produced with the LLE–SPE combination method. However, the single-step LLE extraction yields very low recovery ( 10,000) in full-scan MS mode is sufficient. Because the fragmentation process intrinsically reduces the sensitivity of triple-quadrupole methods, HRMS in full-scan MS mode applications may theoretically improve the detection limits. HRMS instruments also have a much broader mass range compared with the triple-quadruple instruments. While a typical m/z range is 80–1250 for a Sciex API5000, an Exactive-based Orbitrap has a m/z range of 200–4000. Therefore, HRMS is especially advantageous for the analysis of large biopharmaceuticals, such as oligonucleotides. On a benchtop Orbitrap with a quadrupole mass filter, a 1.0 ng/ml LOQ was achievable for an 18-mer phosphorothioate DNA oligonucleotide with 10 µl injection of the neat solution on a 3 µm 2 × 50 Clarity®-RP column, signaling potential applications, at least, in nonregulated environments [Cook K & Bennett P et al., Unpublished Data].AcknowledgementsThe author would like to thank Andrea Jones-Moore and James Fleming from LabCorp for critical review of the manuscript. The author also appreciates Kevin Cook and Patrick Bennett from ThermoScientific for providing relevant HRMS data and discussions.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Tremblay GA, Oldfield PR. Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues. Bioanalysis1(3),595–609 (2009).Link, CAS, Google Scholar2 Efler SM, Zhang L, Noll BO, Uhlmann E, Davis HL. Quantitation of oligodeoxynucleotides in human plasma with a novel hybridization assay offers greatly enhanced sensitivity over capillary electrophoresis. Oligonucleotides15(2),119–131 (2005).Crossref, Medline, CAS, Google Scholar3 Yu RZ, Baker B, Chappell A, Geary RS, Cheung E, Levin AA. Development of an ultrasensitive noncompetitive hybridization ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma. Anal. 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The author also appreciates Kevin Cook and Patrick Bennett from ThermoScientific for providing relevant HRMS data and discussions.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download
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