An Improved Ion-Pair Reversed Phase LC Method for Analysis of Major Impurities of Phosphorothioate Oligonucleotide Cantide
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An improved ion-pair reversed phase LC (IP-RP-LC) method was developed using triethylammonium/hexafluoroisopropanol (TEA/HFIP) buffer as the mobile phase as an alternative to capillary gel electrophoresis (CGE) and ion-exchange chromatography (IEC) for routine quality control of the synthetic 20-mer phosphorothioate oligonucleotide (PS-ODN) Cantide. The most commonly observed impurities included an oligomer with a deletion at the 5′-end (5′n-1) and a monophosphodiester (P=S/O). The method described here was sufficiently selective to simultaneously separate and identify these impurities. To the best of our knowledge, this is the first report on the separation of P=S/O analogs from PS-ODN using IP-RP-LC. Relatively high TEA concentrations and higher pH values were recommended. The separation performance was maintained even in the presence of multiple P=S/O fragments containing different PO residues. The method was successfully applied to the analysis of failure sequences of authentic crude Cantide as well as the purified product. In addition to 5′n-1 and P=S/O analogs, some minor impurities such as 3′n-1 oligomers were also partially resolved.
KeywordsIon-pair chromatography Ion-exchange chromatography Capillary gel electrophoresis Phosphorothioate oligonucleotides Cantide
The authors thank the Beijing Proteome Research Center for their kindly support in the MALDI-TOF-MS analysis. This research was funded by National 863 High-Tech project from the Ministry of Science and Technology of the People’s Republic of China (No. 2007AA021602) and Important National Science and Technology Specific Projects (No. 2009ZX09503-021 and 2009ZX09102-012).
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