Analysis of Oligonucleotides by Size-Exclusion Chromatography: SEC-UV, SEC-MS, SEC-MALS

Jennifer Bouchenna
Claire Butré
Arnaud Delobel

The field of therapeutic oligonucleotides has seen remarkable progress over the last years. Oligonucleotides have emerged as promising candidates for drug therapies for a wide range of diseases. In recent years, the US FDA has approved several oligonucleotide drugs for gene silencing, such as short interfering RNA (siRNA), antisense oligonucleotides (ASOs), microRNA (miRNA) and aptamers. Despite improvement in oligonucleotide synthesis and in post-synthesis clean-up, it is still necessary to monitor oligonucleotide size distribution (impurities such as n-1, n-2, n+1,... oligonucleotides). Furthermore, the presence of multimeric species is also a fundamental aspect of process, quality control and patient safety. Indeed, the presence, or the absence in the case of siRNA, of multimeric species should be verified since it may lead to activity loss of the molecule. 

A large number of techniques exist for the characterisation of therapeutic oligonucleotides (modified or not) in their native state. Indeed, the analysis of oligonucleotide duplexes (and other multiplexes) is a challenge given their susceptibility to dissociation in conventional separation systems that use organic modifiers, ion-pairing agents, and denaturing conditions such as high pH or high temperatures. Size-Exclusion Chromatography (SEC) is particularly well suited for the analysis of these molecules, because separation is based on the use of aqueous buffers under very mild conditions. A good separation is usually achieved due to the large molecular weight differences between the single strand, duplex, and multiplexes and low molecular weight fragments. This application note presents different SEC methods which were developed for the characterisation of oligonucleotides: SEC-UV, SEC-MALS and SEC-MS. . 

Size-Exclusion Chromatography methods with three detection techniques (UV, MALS, MS) were developed for the study of therapeutic oligonucleotides. PBS was used as mobile phase as it provided better results and sharper peaks than other buffers. The method can be coupled to UV as a method of choice for routine analysis to determine the sample purity. Multi-angle light scattering detection should be used to obtain information on the polydispersity of the sample and to identify the multimers that could be present. SEC-MS requires the use of a different mobile phase for compatibility reasons and is used for more detailed characterisation of the samples. With MS, the presence of fragments is indeed detected and the exact molecular mass of the main oligonucleotide, as well as its fragments (n-1, n-2, …), are determined using ammonium acetate as mobile phase. For a separation of n-1 and n-2 impurities, from the monomer, other chromatographic techniques, such as ion pairing reversed phase, should be used. Finally, the use of these three SEC methods is beneficial for the determination of identity and purity of therapeutic oligonucleotides.