Novel strategies of bioanalysis of oligonucleotide therapeutics
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Oligonucleotide therapeutics have emerged as a promising class of drugs to treat a wide range of diseases caused by genetic abnormalities. They have also become indispensable tools for genomic studies allowing for specific knockdown of proteins to study signaling pathways and identify therapeutic targets. Two DNA therapeutics, Fomivirsen and Pegaptanib, have been approved by the US FDA. Replacement of the phosphodiester linkage with a phosphorothioate is one of the most successful modifications made to oligonucleotides to enhance their in vivo stability. The longer elimination phase of phosphorothioates and other modified oligonucleotides requires sensitive and selective methods to quantify the parent drug and their metabolites simultaneously. Traditional hybridization assays are highly sensitive but does not provide selectivity between the parent drug and the metabolites. LC-MS based assay is a promising approach for bioanalysis of oligonucleotide therapeutics due to its selectivity and structure identification capability. However, the lack of sensitivity and complicated sample preparation procedure remains the bottle neck for application of LC-MS based assays to preclinical and clinical studies. Chapter 1 is the introduction and describes the layout of the dissertation. Chapter 2 reviews the literature for analysis of oligonucleotides using LC-MS based methods. Numerous studies have shown that the mobile phase composition has a significant impact on the MS sensitivity of oligonucleotides. In Chapter 3, various ion-pairing agents and counter-ions were evaluated for their effect on electrospray desorption efficiency of oligonucleotides and a novel model of electrospray behavior of oligonucleotides was proposed. The biological sample extraction remains a formidable challenge in developing quantitative LC-MS methods for oligonucleotides. In Chapter 4, a one-step solid phase extraction (SPE) method was developed to retrieve an oligonucleotide from rat plasma. Various issues such as non-specific binding and oxidation of the phosphorothioate linkages were addressed. UHPLC has the potential of greatly increasing the throughput of bioanalysis of oligonucleotide. However, the extensive sample-clean up required for operation under high system pressure and the low flow rate required for sensitive mass spectrometric detection of oligonucleotide poses major hurdles for transferring HPLC-MS methods to UHPLC platforms. In Chapter 5, an ethanol precipitation step was added to the commonly used phenol-chloroform extraction of oligonucleotide and resulted in minimal residual substances in the extracted samples. Other issues such as selecting the optimum organic modifiers, column dimensions, flow rate and scan rate for best chromatographic separation and LC-MS sensitivity of oligonucleotides were also addressed.