Physicochemical property evaluation of modified oligonucleotides by traveling‐wave ion mobility mass spectrometry

Abstract

Rationale Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling‐wave ion mobility mass spectrometry (TWIMS). Methods The physicochemical characteristics of the modified oligonucleotides were determined using IM‐MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross‐section (CCS) values, and CCS widths. Results By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming‐out shape plot at one charge per 4.0–3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS‐modified and unmodified oligonucleotides. The PS‐modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. Conclusions The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry

“…The mobiligram of the 8 mer PS-modified oligonucleotide is composed of 128 diastereomers, and the results show the sum of the separated peaks of these diastereomers. This broader mobiligram tendency has also been reported in previous studies. , …”

“…This broader mobiligram tendency has also been reported in previous studies. 25,26 siRNA Patisiran Impurity Isomer Separation by Cyclic IMS. Based on the above results, we selected the approved full-PO siRNA patisiran sense and antisense strands as models for oligonucleotide isomer separation to confirm the cyclic IMS separation capability.…”

Review of fragmentation of synthetic single‐stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022

“…This transition is observed when around 50% of the molecule is charged, or in other words, when every other phosphate carries a negative charge. Those observations were also studied by Omuro et al 82 and more specifically the effect of the oligonucleotide sequence and length differences on the CCS values by traveling wave ion mobility spectrometry (TWIMS). They observed that when the charge states reached approximately one charge per 3.5–4 bases such as −4 to −5 charge states for 16‐mers, an inflection point was observed, which indicates that the oligonucleotides adopt two conformations in the gas phase, where the conformational change seems to occur around the inflection point.…”

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry

“…For example, in the current LC-UV/MS methodology, impurities with similar masses are simply reported as a single entity (e.g., n-U/n-C or n+U/n+C) with a sum quantity. Recent efforts have been directed toward both LC and MS dimensions to resolve isobaric/isomeric impurity ions. − For example, ion pair-reversed phase (IPRP) LC has been fine-tuned to separate composite impurities and coupled with tandem mass spectrometry (MS/MS) to differentiate unresolved isomers in PO oligonucleotides. , Ion mobility MS has been employed to differentiate PS oligonucleotide diastereomers of short sequences , and to separate isomeric nucleotide residues following nucleic acid digestion. , Nevertheless, many challenges remain, particularly in the context of PS-modified ONTs.…”

“… 11 − 13 For example, ion pair-reversed phase (IPRP) LC has been fine-tuned to separate composite impurities and coupled with tandem mass spectrometry (MS/MS) to differentiate unresolved isomers in PO oligonucleotides. 10 , 15 Ion mobility MS has been employed to differentiate PS oligonucleotide diastereomers of short sequences 16 , 17 and to separate isomeric nucleotide residues following nucleic acid digestion. 11 , 18 Nevertheless, many challenges remain, particularly in the context of PS-modified ONTs.…”