Keys to the Successful Development of Bioanalytical Method for Oligonucleotides

As new modalities in drug development gain popularity, we are in an exciting place to treat and prevent complex disease states. Oligonucleotides or Oligos are heat denatured, short single strand of synthetic DNA or RNA or its analogues such as antisense oligonucleotides (ASOs), small interfering RNAs (SiRNAs), microRNAs (miRNAs), aptamers, synthetic guide RNAs (sgRNAs) and immunostimulatory oligonucleotides. Researchers developing an oligonucleotide must conquer additional challenges compared to a typical small or large molecule therapy, including stability, drug delivery and bioanalytical method development. FDA approved 13 diversified oligonucleotides for several indications since 1998 to treat several diseases. Analytical characterization of oligonucleotides plays an important role in ensuring product integrity and consistency. It is critical to understand these nuances and generate a preclinical strategy to investigate compounds thoroughly. Selection of appropriate analytical techniques depends on the molecular size, higher order structure and chemical composition. To assess the physicochemical and biophysical properties of oligonucleotides, LC-MS/MS assay, hybridized immunoassays and qPCR methods are used. To determine the concentration, pre-treatment steps are critical in extracting oligonucleotides from various type of biological samples. Chromatographic assays, like the liquid chromatography mass spectrometry (LC-MS/MS) method, can help measure an oligo’s concentration in target organs, such as the liver and kidney. These assays can achieve high specificity, accuracy and precision. Typically, a bioanalytical strategy must consider multiple approaches to accomplish DMPK characterization. Method selection should account for the size of the oligonucleotide, sensitivity, matrices, throughput and development stage.