Mobile Phase Aging and Its Impact on Electrospray Ionization of Oligonucleotides.

The implementation of fluoroalcohol/alkylamine mobile phase systems in oligonucleotide LC-MS provides a good balance between chromatographic separations and MS sensitivity. Since its introduction, several parameters including mobile phase composition, additive concentration, alkylamine hydrophobicity, and different fluoroalcohols have been carefully evaluated and optimized. While our understanding of this mobile phase system has increased over the years, there are challenges that continue to hinder method performance and remain poorly understood. One of these challenges is the constant loss of MS sensitivity over time, commonly termed mobile phase aging. This study investigates two aging mechanisms associated with loss of MS sensitivity: alkylamine oxidation and aggregate formation. The relationship between pH, organic solvent, oxygen, and mobile phase aging is characterized, and mitigation strategies to extend mobile phase lifetime are discussed.

Prodrug Strategies for the Development of ß-l-5-((E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU) against Varicella Zoster Virus (VZV).

Varicella zoster virus (VZV) establishes lifelong infection after primary disease and can reactivate. Several drugs are approved to treat VZV diseases, but new antivirals with greater potency are needed. Previously, we identified ß-l-5-((E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU, 1), which had significant anti-VZV activity. In this communication, we report the synthesis and evaluation of numerous l-BHDU prodrugs: amino acid esters (14-26), phosphoramidates (33-34), long-chain lipids (ODE-l-BHDU-MP, 38, and HDP-l-BHDU-MP, 39), and phosphate ester prodrugs (POM-l-BHDU-MP, 41, and POC-l-BHDU-MP, 47). The amino acid ester l-BHDU prodrugs (l-phenylalanine, 16, and l-valine, 17) had a potent antiviral activity with EC50 values of 0.028 and 0.030 µM, respectively. The phosphate ester prodrugs POM-l-BHDU-MP and POC-l-BHDU-MP had a significant anti-VZV activity with EC50 values of 0.035 and 0.034 µM, respectively, and no cellular toxicity (CC50 > 100 µM) was detected. Out of these prodrugs, ODE-l-BHDU-MP (38) and POM-l-BHDU-MP (41) were selected for further evaluation in future studies.

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

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.