Isolating and Analyzing Therapeutic Oligonucleotides from Biological Matrices

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Figure 1. Overlaid UV chromatograms of a oligonucleotide extracted from plasma using the Clarity OTX protocol compared to an equal amount of an oligonucleotide standard directly injected on HPLC. Note the near quantitative recovery of the oligonucleotide from plasma using Clarity along with the lack of any interfering plasma components. (Source: Phenomenex)

There has been a recent explosion of oligonucleotide therapeutics entering animal and clinical trials and drug developers are interested in their pharmacokinetic properties. Isolating and analyzing oligonucleotides from biological matrices is the major obstacle in developing these therapeutics. Oligonucleotides are acidic and highly polar, which make them difficult to isolate from other plasma components for LC/MS analysis. There is a manually intensive method using liquid/liquid extraction combined with SPE to isolate oligonucleotides from biological fluids; however, that method is not practical for a clinical trial where hundreds of samples may need to be analyzed.

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Figure 2. LC/MS chromatograms of different levels of oligonucleotide extracted from liver tissue using Clarity OTX. Note the good linearity for oligonucleotide extracted from a tissue sample. (Source: Phenomenex)

To fulfill this unmet need, a single-step sample preparation technique was developed using Phenomenex Clarity OTX cartridges. Serum or plasma containing a synthetic oligonucleotide is treated with solubilization buffer and then loaded on the Clarity OTX cartridge. The cartridge is washed with organic to remove proteins and lipids that could interfere with mass spectroscopy (MS), then eluted with a different buffered organic solution. The whole procedure takes less than 15 minutes and is easily automated and multiplexed in a 96-well plate.

Extracted oligos and their metabolites are separated using a core-shell Clarity Oligo-MS HPLC column and identified by MS using ProMass deconvolution software. Figure 1 gives an example chromatogram of a therapeutic oligonucleotide extracted from plasma and overlaid with an equal amount of a control oligonucleotide. The extraction protocol using Clarity OTX generally delivers recoveries greater than 80% with minimal MS-interfering contaminants. When sensitive MS instrumentation is used, quantitation has been observed well into the low picomolar range. The methodology has been expanded to allow extraction of oligonucleotides from tissue samples in concert with the use of common cell homogenization techniques. In Figure 2 a different oligonucleotide is recovered from a liver sample with only a slight increase in background contaminants. This new extraction and analysis protocol allows for rapid isolation and analysis of the large numbers of samples expected from a clinical trial. This methodology is a serious paradigm change in the development pipeline of oligonucleotide therapeutics.

This article was published in Drug Discovery & Development magazine: Vol. 13, No. 3, April 2010, p. 26.