Optimising Liquid Phase Oligo Synthesis Detritylation

In an effort to counteract the scalability issues seen with traditional solid phase oligonucleotide synthesis, AstraZeneca are currently investigating opportunities to implement liquid phase methods. This presentation specifically focuses on the detritylation process, one of the three steps in AstraZeneca’s oligo synthesis. 

Boulter began by sharing a chromatogram which detailed her team’s first attempt at synthesising an AstraZeneca IP. This first attempt had a poor yield at just 3.3% and so required lots of improvement. However, the team were hopeful, as they were still successful in synthesising some product. Therefore, Boulter’s team went ahead in improving product purity, and to develop a robust and consistent detritylation. 

Tackling detritylation, Boulter took a kinetic modelling approach to investigate the impurities. Using this approach, the team analysed the impact of various factors, such as temperature and acid concentration, on the detritylation process. It allowed for significant improvements in yields and process understanding. 

AstraZeneca use co solvents along with the traditional acetonitrile. Boulter experimentally tested the impact of these solvents on reaction rates and found that benzonitrile was able to accelerate the rate by almost five times. Furthermore, investigating nucleobase variation, the team hypothesized that pKa differences caused differences in acid concentration to reaction rate relationship. This was then incorporated into their kinetic model. 

They also introduced inline Raman spectroscopy to better understand the full complexity of the detritylation process. Using Raman, they were able to monitor the detritylation process in real-time. This technology enabled precise reaction endpoint predictions and enhanced process control. 

At the end of the presentation, Boulter shared a chromatogram of their fourteenth liquid-phase synthesis run, after implementing all the optimisation steps. Compared to the first run, product purity showed a dramatic improvement. Purity increased over twenty times from 3.3% to 70%. Boulter emphasised the potential for these advancements to streamline future oligonucleotide syntheses, reducing time from 18 months to just weeks and increasing sustainability through higher yields and reduced process mass intensity.