Stephen Luke Product Manager Agilent Technologies

Synthetic oligonucleotides have emerged as promising therapeutic agents for the treatment of a variety of diseases, including viral infections and cancer. Researchers are looking at several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and aptamers, for therapeutic applications.

However, various impurities – product-related, in the starting materials, and arising from incomplete capping of coupling reactions – must be identified and removed and postsynthesis processing must be monitored. Thus, a key challenge in the development and manufacture of oligonucleotide therapeutics is to establish analytical methods that are capable of separating and identifying impurities.


Exploring Better Options for Oligonucleotide LC Separations

Ion-pair, reversed-phase separation of the trityl-on oligos and is relatively simple to perform. This method separates the full-length target oligo, which still has the dMT group attached, from the deprotected failure sequences. The analytical information obtained is limited, so this is generally considered a purification method.

An alternate method, ion-exchange separations of the trityl-off, deprotected oligos uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column, making the technique unsuitable for use with LC/MS.

Finally, ion-pair, reversed-phase separation of the trityl-off, deprotected oligos makes use of organic solvents and mobile phase additives such as TEAA (triethylammonium acetate) or TEA-HFIP (triethylamine and hexafluoroisopropanol) to ion-pair with the negatively charged phosphodiester backbone of the oligonucleotide. High-performance columns deliver excellent resolution. What’s more, methods with volatile mobile phase constituents such as TEA-HFIP are suitable for use with LC/MS, providing useful information to help characterize oligonucleotide structures and sequences.

In Table 1 we summarize some of the options for oligonucleotide analysis by liquid chromatography.

Designed for ion-pair, reversed-phase separation of the trityl-off, deprotected oligos using either TEAA or TEA-HFIP mobile phases –Agilent AdvanceBio Oligonucleotide columns meet these challenges.


Table 1. Options for oligonucleotide LC separations

Addressing the Need for Better Resolution and Increased Lifetime

Successful ion-pair reversed-phase separation of the trityl-off, deprotected oligos requires columns that have high resolving power and are robust enough to withstand the relatively aggressive analysis conditions. Without sufficient resolution, the accuracy and precision of measurements can be compromised, leading to a lack of confidence in the analytical results. Columns that are not robust will have a short lifetime, resulting in frequent replacement, disruption to workflows, and increased costs.


Agilent Oligonucleotide Resolution standard for peak performance

Agilent AdvanceBio Oligonucleotide columns feature high-efficiency, 2.7 µm superficially porous Poroshell particles. The particles are chemically modified using proprietary technology that makes them very resistant to high pH mobile phases. They are bonded with an endcapped C18 phase to deliver excellent selectivity for oligonucleotides. To ensure performance for your separations, every batch of AdvanceBio Oligonucleotide media is tested with an Agilent Oligonucleotide Resolution standard.

The ability to resolve oligonucleotides that differ by a single nucleotide is important for accurate characterization. Figure 1 shows how the AdvanceBio Oligonucleotide, 2.1 x 50 mm column resolves N/N-1 oligonucleotides, with sharp peaks and good selectivity.


Figure 1. The Agilent AdvanceBio Oligonucleotide column gives sharp peaks and high resolution for N/N-1 oligonucleotides.

Achieving Consistent Results – With Column Stability

Column stability is another important feature of the Agilent AdvanceBio Oligonucleotide column. Figure 2 shows how the AdvanceBio Oligonucleotide column remains stable over about 400 injections using TEAA mobile phase.


Characterizing Oligonucleotide Structures and Sequences Using MS Compatibility

LC/MS provides useful information to help characterize oligonucleotide structures and sequences. The AdvanceBio Oligonucleotide column gives high chromatographic resolution and MS sensitivity using HFIP-TEA mobile phase. The Agilent AdvanceBio Oligonucleotide column with accurate mass MS characterizes oligonucleotide structures and sequences, as shown in Table 2 and Figure 3.


























Agilent Related Content:
ON-DEMAND WEBINAR: Accurate and reliable characterization of impurities in oligonucleotide therapeutics

Stephen Luke is product manager, reversed-phase biocolumns at Agilent Technologies.

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