Silence Therapeutics works on double stranded siRNA molecules, a hybridisation of both antisense and sense strands. Both of these strands are obtained by solid phase synthesis before being heavily modified to increase their biological activity and enzymatic stability. 

These include ribose modifications like methoxy and fluoro modifications and an alternating pattern on the antisense strand. Silence introduces four phosphorothioate groups on the antisense strand, and six on the sense strand. Furthermore, the sense strand is conjugated to a GalNAc-cluster, which is also phosphothioated to direct delivery to the liver. 

Solid phase synthesis of these products results in several product-related impurities which have a structure similar to the parental sequence. This presentation focuses on the most common impurities, including:  

  • N-1 shortmer impurities: the same sequence but missing one nucleotide at the 3’ and 5’ ends. These form during coupling or detritylation steps. 

  • N+1 longmer impurities: resulting from the insertion of one or more nucleotides, also formed during coupling. 

  • PS→PO impurities: these relate to phosphodiester where phosphorothioate is missing at the 3’ or 5’ end. These form during sulfurisation or storage. 

  • GalNAc impurities: where the sequence is missing one or more GalNAc moieties. These are only related to the sense strand. 

How are these impurities characterised? Silence mainly employs two liquid chromatography-based analytical tools: ion pair reverse phase liquid chromatography (IP-RP-LC) and anion exchange liquid chromatography (AEX-LC). These both have the advantage of being widely used and therefore well-studied; as such, the parameters are well understood. 

However, these tools also have several disadvantages. AEX-LC cannot be coupled to the mass-detector, so the reading only indicates purity and not mass. IP-RP-LC can be coupled to a mass-detector, but it relies on unstable, toxic and expensive mobile phase additives. Due to these problems and tightening regulations for oligonucleotides, a wider range of analytical methods is needed. 

That’s where hydrophilic interaction liquid chromatography (HELIC) comes in. This emerging analytical tool offers mass compatibility with stable, nontoxic, and cheap mobile phase additives. 

The rest of the presentation describes the development of an analytical method using HILIC, including optimising parameters such as stationary phase, mobile phase composition, and temperature, leading to successful separation of oligonucleotides. The lab aims to improve mass sensitivity in HILIC and is working on characterising phosphorothioated oligonucleotides and double-stranded molecules.