Oligonucleotides are a rising class of drugs which have entered the market as biopharmaceuticals due to their ability to interfere directly with gene expression instead of targeting the protein. The production of oligonucleotides is usually conducted by chemical solid-phase synthesis and use of nucleoside phosphoramidites as building blocks. To prevent undesired side reactions, protecting groups are attached to the nucleosides, for example the 5’-hydroxy group of the sugar moiety is protected with a dimethoxytrityl (DMT) group.
During oligonucleotide synthesis errors accumulate so that impurities and degradation products emerge. The major challenge in oligonucleotide purification is the presence of structurally similar impurities, such as shortmers (n–x) and longmers (n+x), which are hard to separate from the desired product. Therefore, analysis of synthesised oligonucleotides by analytical chromatography is vital to monitor their quality and quantity before and after purification. A robust set-up is needed to achieve reliable and highly reproducible results.
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