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

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.

Evaluation of alkylamines and stationary phases to improve LC-MS of oligonucleotides.

This study evaluated four bridged-ethylene hybrid (BEH) columns containing C18 (130 Å), peptide C18 (300 Å), phenyl, or a mixed-mode charged surface hybrid (CSH C18 ) using a wide range of antisense oligonucleotide therapeutics. The BEH C18 , peptide, and phenyl columns were all capable of providing significant retention of oligonucleotide samples across multiple ion-pairing systems using alkylamines and 1,1,1,3,3,3,-hexafluoroisopropanol (HFIP). The retention of the oligonucleotides varied depending on the choice of alkylamine, with the order of retention being dimethylcyclohexylamine > diisopropylethylamine > triethylamine. The selectivity of these columns for several closely eluting impurities was similar. Although overall the C18 , peptide, and phenyl columns were all found to be capable of analyzing oligonucleotide therapeutics, the phenyl column was found to be the most retentive and the C18 column provided the best peak shape. The CSH C18 column was found to be degraded by the alkylamine-HFIP mobile phase despite the mobile phase being within the pH stability range of the column.