Bioanalytical LC–MS of therapeutic oligonucleotides
Therapeutic oligonucleotides (OGNTs) are important biopharmaceutical drugs for the future, because of their ability to selectively reduce or knock-out the expression of target genes. For the development of OGNTs, reliable and relatively high-throughput bioanalytical methods are required to perform the quantitative bioanalysis of OGNTs and their metabolites in biological fluids (plasma, urine, tissue, etc.). Although immunoaffinity methods, especially ELISA, are currently widely applied for this purpose, the potential of liquid chromatography coupled to mass spectrometry (LC–MS) in OGNT analysis is under investigation. Because of its inherent ability to monitor the individual target OGNTs as well as their metabolites, LC–MS is now evolving into the method-of-choice for the bioanalysis of OGNTs. In this paper, the state-of-the-art of bioanalytical LC–MS of OGNTs and their metabolites in biological fluids (plasma, urine, tissue, etc.) is critically reviewed and its advantages and limitations are highlighted. Finally, the future perspective of bioanalytical LC–MS, i.e., lower detection levels and potential generic LC–MS methodology, is discussed.
At present, short-chain oligomers are applied and under investigation as oligonucleotide therapeutics (OGNTs), typically between 15 and 50 nucleotide units long. These OGNTs are built to interfere with the processing of genetic information by acting on the DNA or RNA and thereby interfere with the production of (disease-related) proteins in the cell (1). Typical examples of OGNTs are Vitravene to treat cytomegalovirus retinitis in AIDS patients and Macugen to treat age-related macular degeneration. Other OGNTs are currently under development for the treatment of cancers, diabetes, ALS, Duchenne muscular dystrophy and diseases such as asthma and arthritis with an inflammatory component.
Antisense oligonucleotides (asRNAs) are currently the most successful representatives of the first generation OGNTs. The asRNA hybridizes to a specific sequence of the mRNA, thereby interfering with normal translation and inhibiting or decreasing expression of the target protein (2). Another type of OGNTs is based on a naturally occurring gene silencing mechanism called RNA interference (RNAi), where short double-stranded RNAs knock down gene expressio ...