Ken Cook Ph.D. EU Bio-Separations Expert Thermo Fisher Scientific

This is the third article in this series discussing HPLC oligonucleotide separations.  Anion exchange chromatography can provide additional information when characterizing oligonucleotides. It can be linked in an orthogonal workflow with IPRP and interfaced with MS using an efficient automated desalting technique.

There is a natural migration in the use Ion Pair Reversed-phase (IPRP) for Oligonucleotide [ON] characterisation when the ability of interfacing with mass spectroscopy [MS] is required. However, several classes of isobaric impurities may need further separation before MS and there are many HPLC only separation methods where ion exchange proves to be the better option. In addition, the Thermo Scientific™ DNAPac™PA200 which is the market gold standard is now available in a UHPLC 4µm particle size version, keeping pace with UHPLC separation efficiencies. In this article we discus some of the high resolution characterisation that can be delivered by ion exchange and how it can be used as an additional orthogonal method combined with IPRP. We will also show good options on how to interface with MS.

The most common analysis for oligonucleotides is the N-X sequence failures. This is the production of a small amount of short length oligonucleotide due to a failed addition of one of the nucleotides during synthesis. This analysis can be done equally well with ion exchange or IPRP and has been shown before in the first article of this series, revealing the different selectivity obtained with each technique.

Separation of identical length oligonucleotides is possible with ion exchange and such separations can be modulated with control of the pH.  Between pH 8 and 10.5 ionization of Thymine (T) and Guanine (G)  [uridine in RNA] will occur producing extra negative charges which in turn will increase the retention time on ion exchange. This is a good technique to exploit differences in the base content of the ON and the possibilities are shown in figure 1. This technique is not possible on silica based columns due to the pH restrictions on these resins.

Figure 1

Another common analytical requirement is to look at the stability and the formulation of double stranded therapeutic oligonucleotides. The sense and the corresponding antisense strands would be synthesized, purified and annealed to form a specific duplex therapeutic oligonucleotide. The short dsRNA species allows targeted control of the expression of proteins involved at the route of many disease states. Anion-exchange analyses of double-stranded (ds) and Single-Stranded (ss) oligonucleotides have been used in stability-indicating methods. These are required for assessment of sense and antisense formulations to verify correct formulation and stability during storage. The use of elevated temperatures to produce fully- or partially-denaturing conditions is a common technique and can be used with ion exchange separations. As opposed to reverse phase separations, increased temperature with anion exchange separations will increase retention allowing better optimization of ss and duplex ONs. Figure 2 shows a separation produced at 70C which clearly shows a slight excess of the sense RNA strand in the formulation which has been analysed.

Figure 2

Therapeutic oligonucleotides often include modifications to increase their biological half-life. One of the most common is the introduction of phosphorothioate (PS) linkages to restrict the susceptibility of the therapeutic ON to nuclease activity. Complete thiolation needs to be confirmed and ion exchange analysis has proven useful in this application. When the thiolation has not gone to completion an unwanted PO linkage will be present instead of a PS. One of the problems with this analysis is that the PO impurity elutes before the main peak and can be difficult to separate from the N-1 impurities also present. Using a 2 dimensional approach with IPRP / Ion Exchange and opting for a size only selectivity method in IPRP we can circumvent this problem by first removing the N-X impurities before looking for the PO impurities in the ion exchange run. This is an example of adjusting the selectivity of the columns coupled with the inherent selectivity differences between ion exchange and IPRP. This is shown in figure 3 where a siRNA sample was heavily loaded for fractionation on an IPRP column using a TBAB eluent to select for a size separation. The collected main peak, now free from N-X impurities was then subjected to anion exchange to identify the PO 

Figure 3

It is possible to link ion exchange to MS where anion exchange separations are preferred.  This is achieved by trapping the ion exchange separated ON’s onto a reverse phase trap column for salt removal with an MS-compatible solvent such as acetonitrile using ammonium formate as a week ion pair. This approach has been found to be much faster and more effective at salt removal than dialysis or size exclusion chromatography. It also supports the selection of eluents without ion-suppression, affording improved MS sensitivity. The De-convoluted spectra in Figure 4 of a synthetic single stranded DNA is fairly clean, showing only a small amount of a single sodium adduct left in the sample following the desalting process.

The examples shown are only a few of the high resolution separations of oligonucleotides that are possible using an anion exchange approach. The different selectivity provides an additional approach for these characterizations to IPRP.  Additional analytical methods provided by anion exchange include confirmation of 2´-5´ aberrant linkages, resolution of PS-diasteroisomers and Extraction of ON’s from biological fluids and tissues. Anion-exchange chromatography often delivers improved Antisense and sense strand analyses and can contribute in difficult PS / PO separations. The ability to rapidly desalt fractions collected from high resolution ion exchangers also provides compatibility with MS for identity confirmation. Any laboratory looking to fully characterize oligonucleotides will require both IP-RPLC and high resolution anion exchange capabilities.

Figure 4

Additional Resources:

A. DNAPac PA200 Product Specifications

B. Application Note: High-Resolution Separation of Oligonucleotides on a Pellicular Anion-Exchange Column

C. Poster Note: High-speed, High-resolution Oligonucleotide Separations Using Small Particle Anion-Exchangers

D. Application Note: Ultra-High-Resolution Separation of Oligonucleotides on Pellicular Anion-Exchange UHPLC Columns

Ken Cook, Ph.D. ([email protected]), is EU Bio-Separations Expert at Thermo Fisher Scientific.

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