26 May / June 2017
Chromatography for the Analysis of Oligonucleotides
by Julia Baek, Jim Thayer, Shanhua Lin, Ilze Birznieks, Kyle D’Silva, Simon Cubbon Biopharmaceutical Characterization Center of Excellence, Thermo Fisher Scientific, 490 Lakeside Dr, Sunnyvale, CA 94085, USA
With increasing medicinal applications of therapeutic oligonucleotides, and several candidates currently in clinical trials, there is the need to perform analytical characterisation of these drugs, which can be challenging. Two high-resolution chromatographic options for this analysis are the use of either non-porous anion exchange (AEX) or porous ion pair reverse phase (IPRP) UHPLC. Both approaches have their own advantages and also offer differences in selectivities. Here we present chromatographic methodologies for the separation of;
• single stranded DNA, • failure sequences, • fluorescent dye-labelled DNA, • large double-stranded DNA fragments.
Introduction
Synthetic oligonucleotide therapeutics are widely used for a variety of applications including DNA amplification and sequencing, in-situ hybridisation, gene silencing and molecular diagnostics. These drugs span a range of oligonucleotide classes, indications, and routes of administration. Synthetic oligonucleotides have motivated drug developers with the promise of rational drug design, lower drug development costs, and the ability to reach targets that conventional small molecule drugs cannot. They also have now emerged as promising therapeutic candidates for diseases including cancer, viral infections, Alzheimer’s disease and cardiovascular disorders. Many therapeutic oligonucleotides, including antisense, aptamers and small-interfering RNAs (siRNAs) are being developed. This has created an increased demand for the analysis of these synthetic nucleotide products beyond previous requirements.
The oligonucleotide field is approaching a critical turning point with numerous therapeutics in phase 3 clinical trials. Within the next few years, regulatory approval of multiple oligonucleotide therapeutics seems likely [1]. New start-up companies with novel technologies will likely continue to propel the field forward and fuel the development pipeline with new therapeutic options.
Oligonucleotides for molecular and therapeutic applications demand high resolution purity analysis, as well as identification and quantification of any structural impurities. Therefore, quality control of these synthetic oligonucleotides is critical, requiring stable, high resolution tools to deliver the analytical evaluations required.
siRNA can be used to control translation, and hence the amount of specific target proteins that contribute to disease states. RNA aptamers can have very high binding affinities to specific targets, essentially making them the oligonucleotide analogue of a monoclonal antibody. To be effective in the body these oligonucleotides must be resistant to endogenous nuclease attack that would quickly degrade them. To achieve this, they can be chemically modified by thiolation of the backbone to resist enzymatic breakdown and produce a therapeutically viable biological half- life, which generates further demands on the analytical characterisation of these molecules.
Options for Characterisation
With the increase in medicinal applications, the analytical characterisation required by the regulatory bodies has become more stringent. The higher resolution
options for this analysis have proven to be either non-porous anion exchange (AEX) or ion-pair reversed phase (IPRP) UHPLC. Both have their own advantages and differences in selectivities. Because of this, many laboratories employ the use of both techniques to ensure complete characterisation. IPRP has the advantage of direct coupling to mass spectroscopy (MS) [1]. In this methodology overview, we present:
• Fast, High Resolution Separation of Single Stranded DNA
• Separation of Failure Sequences
• Separation of Fluorescent Dye-Labelled DNA
• Separation of Large Double-Stranded DNA Fragments
Fast, High Resolution Analysis of ss- and ds-Nucleic Acids using Reversed-phase UHPLC
Synthetic oligonucleotides are used extensively in laboratories as primers for polymerase chain reactions (PCR) and DNA sequencing, probes to visualise a specific DNA or RNA, tools to study gene function, and biopharmaceutical drugs for treating various diseases [1,2]. Analyses of synthetic oligonucleotides are most commonly performed using ion-pair reversed-phase
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