(M1330-06-31) Optimising the Extraction of Oligonucleotides by SPE

Purpose: The use of oligonucleotides and nucleic acids as therapeutics is becoming more pronounced, and the recent Covid-19 pandemic has demonstrated the advantages that these new modalities can have over small molecule drugs. However, this has started to present the bioanalyst with a significant challenge as oligonucleotides are very complex molecules and the resulting chromatography and extraction protocols have to be optimised to ensure that this level of complexity is addressed. An understanding of the extraction process and how to optimise it is critical, as is using the correct type of extraction media. This poster examines each stage of extraction and demonstrates the use of a novel composited silica based SPE media to improve the robustness of the final assay.

Methods: An LC-MS method was initially developed for a test oligonucleotide, this was then used to analyse a series of samples derived from the loading of an aqueous oligonucleotide. Each stage of the solid phase extraction protocol was investigated to assess the impact that it had on the recovery of the overall assay. In particular the use of ion pairing and organic solvents was investigated, as well as the use of multiple elutions to optimise the extraction process when using an anion exchange SPE media. A simple molecular modelling package, Chemicalize.org, was utilised to support the experimental findings to aid a more generic approach to method development to be attained.

Results: The experimental and molecular modelling data demonstrated that the use of organic solvents greatly reduced the effectiveness of the extraction process. The modelling work used a log D predictor based on a single nucleotide. The predicted log D was highly negative across the entire pH, suggesting that solubility in an organic solvent would be problematic, but the number of pK_as present, even with just one nucleotide, gave insights into some of the issues that bioanalysts would have analysing such compounds, compared to traditional small molecules. It was determined that the oligonucleotides were not so soluble in methanol, which led to an interesting observation on the ion exchange media that the optimal elution conditions were water, modified by an ion pairing reagent. Experimentally, the use of multiple elutions and the optimisation of the total elution volume was also deemed to be an important experimental parameter to optimise, and there is data to support this statement. During the method development stage, a novel composite SPE media was employed and this was found to have much better recovery and useability when applied to the analysis of biological samples, with recoveries at values greater than 80% for the composite material compared to ca. 60% for loose packed media. Traditional loose packed SPE media was shown to have issues with reproducibility, in particular, where individual samples would fail, jeopardising the whole batch of samples being analysed.

Conclusion: The work presented in this poster examines each stage of the SPE extraction of oligonucleotides to develop an understanding of the impact of critical parameters on analyte recovery. A combination of molecular modelling and experimental data were assessed. Optimisation of elution solvent, ion pairing reagent and elution volume were found to be critical parts of the protocol to optimise. The use of a novel composite SPE media was found to provide improved recovery and reproducibility over loose packed materials.