Current State of Oligonucleotide Characterization Using Liquid Chromatography–Mass Spectrometry: Insight into Critical Issues

Abstract

As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.

Solid-Phase Synthesis of Boranophosphate/Phosphorothioate/Phosphate Chimeric Oligonucleotides and Their Potential as Antisense Oligonucleotides

“…It is worth noting that although we had purified PB containing ODNs by anion-exchange HPLC, it was found that RP-HPLC purification using a mixture of methanol and a buffer containing hexafluoroisopropanol and triethylamine as an eluent was effective for isolation of these ODNs. 47 Thus, all of the ODNs but 22 were purified by RP-HPLC. Although there is room for further improvement of yields, some kinds of chimeric ODNs were successfully synthesized in the strategy.…”

Tandem mass spectrometric sequence characterization of synthetic thymidine‐rich oligonucleotides

“…For this work, hydrophilic interaction liquid chromatography (HILIC), which uses MS compatible mobile phases, was used as an alternative to more widely used ion-pair reversed-phase liquid chromatography (IPRP LC) to separate oligonucleotides prior to MS analysis. 6,7,11,[20][21][22] The absence of ion-pairing (IP) reagents during HILIC-MS analysis helps address some of the IP reagents-related technical hurdles, including The results demonstrated a good peak shape and an envelope of charge state distribution centered on the À4 charge state (Figure 1 inset), which was different from the wide charge state distributions commonly observed during IPRP LC-MS analysis of oligonucleotides. 20 Importantly, a predominant charge state offers better analytical sensitivity by consolidating the molecular ion count and is beneficial when using an extracted ion chromatogram for quantitation or when selecting a precursor ion in MS/MS.…”

“…5,14,15 In addition, structural modifications of oligonucleotides, such as internucleotide phosphodiester linkages replaced by phosphorothioate (PS) linkages, may pose potential challenges to the sequence elucidation by MS/MS because of the added resistance to bond cleavages by the modified linkages. 7,8,13,14,16 In this study, sequence characterization of custom synthesized DNA oligonucleotides of the same nucleotide sequence as nusinersen (an 18-mer with seven T units, marketed as Spinraza™ and approved by FDA in 2016) with or without PS modification was investigated using two ion activation techniques: resonant or trap-type collisioninduced dissociation ("CID") and beam-type CID or higher-energy collisional dissociation ("HCD"). 15,17,18 The feasibility and suitability of MS/MS fragmentation by CID or HCD were compared for the ability to produce diagnostic fragment ions for the sequence characterization of T-rich PS-modified oligonucleotides.…”

“…Thymidine (T)‐rich oligonucleotides are notoriously difficult to sequence because of unfavorable bond cleavages at thymidine residues by MS/MS 5,14,15 . In addition, structural modifications of oligonucleotides, such as inter‐nucleotide phosphodiester linkages replaced by phosphorothioate (PS) linkages, may pose potential challenges to the sequence elucidation by MS/MS because of the added resistance to bond cleavages by the modified linkages 7,8,13,14,16 …”

“…MS/MS of DNA (unmodified): CID versus HCDSequence characterization of oligonucleotides by MS/MS fragmentation of multiply charged oligonucleotide precursor ions in negative ion mode has been intensively studied 5,7. Four possible cleavage sites along the phosphodiester backbone through corresponding dissociation channels can produce four series of sequence-defining complementary ion pairs.…”