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Counting on Faster Flow Cytometry
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UHPLC Methodologies for the Analysis of Oligonucleotides
This is the first article in a series, describing the characterization of oligonucleotides using both Ion Exchange and Ion Pair Reverse Phase HPLC. This article looks at the benefits of each technique and how to tailor the separations to suit the application requirements.
Synthetic oligonucleotides have been widely used for various applications including DNA amplification and sequencing, in situ hybridization, gene silencing and molecular diagnostics. They have now also 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 currently in clinical trials. This has created an increased demand for the analysis of these synthetic nucleotide products beyond the previous requirements. Oligonucleotides for molecular and therapeutic applications require high resolution purity analysis, as well as identification and quantification of their structural impurities. Therefore quality control of these synthetic oligonucleotides is important, so stable, high resolution tools for these analytical evaluations are needed.
There is a growing investment in therapeutic oligonucleotides by the pharmaceutical companies. Small interfering RNA [siRNA] can be used to control the 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 nucleosidase 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. This generates further demands on the analytical characterisation of these molecules.
Options for CharacterisationClick Image To Enlarge +Figure 1 shows an example of the difference in selectivity between Anion exchange and IPRP.
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 reverse phase [IPRP] UHPLC. Both have their own advantages and differences in selectivity’s. 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]. However there is a high level of an ion pair such as Tri Ethyl Ammonium Acetate [TEAA] used which effectively converts the reverse phase column into a pseudo-ion exchange column. This amount of TEAA used causes ion suppression and usually contaminates the LCMS system with the ion pair reagent, effectively limiting the use of the instrument to negative ion mode until it is cleaned. Subsequent improvements to this method employed the use of hexafluoroisopropanol [HFIP] with lower levels of ion pair, reducing the ion suppression and increasing sensitivity when coupling to MS. The Thermo Scientific™ DNAPac™ anion exchange columns have proven to be the gold standard for nucleotide separations and there is now a UHPLC version of this column. A high level of salt is used in the eluent system which prevents direct coupling to MS and the corrosive nature of the eluent requires the use of an inert UHPLC system to ensure robustness of the method. Ion exchange can be used in conjunction with MS if a suitable desalting method is employed and this can be automated in a 2 dimensional approach. Trapping onto a reverse phase cartridge using acetonitrile and ammonium formate as an eluent system has proven to be very fast an effective way to remove salt and limit both adduction and ion suppression, resulting in improved MS sensitivity. While MS can confirm base composition and answer other questions in oligonucleotide characterisation, there are some modifications which result in isobaric products that the MS system cannot discriminate. In such cases as incorrect linkages there must be a suitable UHPLC separation. There are many methods in quality control where the UHPLC assay provides enough selectivity to stand on its own. Whichever LC method proves more favourable for the analysis the availability of both techniques provides the option for a more thorough characterisation.
The Anion exchange column used was a Thermo Scientific™ DNAPac™ PA200 RS using a sodium chloride eluent system and the IPRP column was a polymeric Thermo Scientific™ DNAPac™ RP with a TEAA eluent system. The sample was a single stranded synthetic DNA and both separations are similar but differences in the selectivity can still be seen. The separation mechanism for both techniques addresses primarily size then sequence or base specificity to a lesser degree. These methods can be tailored using different eluent systems to favour separations based mainly on size or to introduce greater sequence specificity. Perchlorate eluent systems in anion exchange produce separations based mainly on size, where sodium chloride eluents provide more selectivity for resolution of nucleotide sequence and modification variations. With IPRP an eluent system based solely on TEAA allows sequence differences to be exploited. Addition of HFIP or use of hexylamonium acetate will convert this to size only as will the use of tri butyl ammonium acetate. In addition, the pH can be raised to between 9-11 on polymeric resins. This causes the ionisation of the tautomeric oxygen present only on guanine and thymine (uridine in RNA) bases. The additional negative charge will increase retention of the oligonucleotide. This gives additional sequence separation depending on the amount of these two bases present in the sequence. This separation power was restricted to the polymeric anion exchange resins due to the pH limitations of silica reverse phase columns. However there is now a polymeric UHPLC reverse phase column available in the Thermo Scientific™ DNAPac™ range for oligonucleotide separations where this option has proven effective.