ABSTRACT
Solid phase synthesis of oligonucleotides requires stepwise addition of nucleotides to a growing chain. Although this process is highly ef‰cient, failure sequences and process related modi‰cations and impurities are often present in the ‰nal product. Depending on the application, particularly
4.1 Introduction .......................................................................................................................... 137 4.2 Background ........................................................................................................................... 138 4.3 Chromatographic Considerations for MS Analysis .............................................................. 139
4.3.1 Effect of the Solution pH .......................................................................................... 140 4.3.2 Ion-Pairing Reagent .................................................................................................. 141 4.3.3 Desalting ................................................................................................................... 146
4.4 Molecular Weight Determination ......................................................................................... 147 4.5 Quantitative LC-MS ............................................................................................................. 149 4.6 Common Impurities .............................................................................................................. 151
4.6.1 Shortmers .................................................................................................................. 151 4.6.2 Longmers .................................................................................................................. 152 4.6.3 Incomplete Removal of Protecting Groups .............................................................. 152 4.6.4 Acid Treatment Related Impurities........................................................................... 153 4.6.5 Base Treatment Related Impurities .......................................................................... 153 4.6.6 Oligonucleotide Degradation Products ..................................................................... 153
4.7 Applications .......................................................................................................................... 154 4.7.1 High-Throughput Desalting Analysis of Oligonucleotides ...................................... 154 4.7.2 Sequence Con‰rmation by LC-MS .......................................................................... 156 4.7.3 Analysis of Phosphorothioates.................................................................................. 156 4.7.4 Analysis of Duplex Oligonucleotides ....................................................................... 157 4.7.5 Aptamers ................................................................................................................... 161 4.7.6 Oligonucleotide Bioanalysis ..................................................................................... 162
4.8 Conclusion ............................................................................................................................ 162 References ...................................................................................................................................... 163
when these sequences are intended for use in therapeutic applications, identi‰cation of impurities is important for the optimization of the synthetic process. With respect to therapeutic applications, impurities can have toxicological implications; therefore, their identi‰cation and minimization in therapeutic oligonucleotides is important.1
