Oligonucleotides, typically 18 to 25 nucleotides in length, are difficult to prepare at large scale and high purity using solid phase synthesis (SPS) of oligonucleotides, thereby requiring the use of extensive chromatography downstream to reach acceptable quality for clinical applications. The author has developed and optimised a liquid phase synthesis (LPS) methodology for preparing oligonucleotides that intrinsically allowed on-line monitoring of the coupling and deprotection reactions through UPLC-MS to be observed, and their respective kinetics and efficiencies throughout the synthesis to be determined. Thereby, any incomplete reactions were detected and driven to completion to improve synthesis yield and purity. This novel reaction monitoring system (RMS) enabled the selection of reagents and solvents that increased the reaction rate of both the coupling and detritylation reactions. This was in lieu of preventing side reactions caused by slow reaction kinetics.
After each reaction, the growing oligonucleotide was separated from reaction debris using a two-stage organic solvent nanofiltration (OSN) process. To enhance the separation, three independently growing oligonucleotides were linked to a central soluble hub molecule to create a 3-arm oligo homostar. This specific diafiltration process was named nanostar sieving (NS). The purified oligo homostar is then concentrated, again using membrane filtration, ready for the next chain extension. This methodology enabled LPS to be integrated into a closed loop process. Process engineering development led to the creation of synthesisers at 10-gram scale with process scale models built to represent 10-kilogram synthesisers based off experimental data collected, compared against process mass intensity (PMI) and economic data through a workshare agreement with GSK.
The necessity for pure and dehydrated solvents, which was investigated through packed bed adsorption of water using zeolite molecular sieves, included adsorption isotherm data and the development of a process model to predict solvent dehydration. This research has introduced a disruptive technology platform, to compete against the current industry standard SPS and ultimately aimed at instilling change in current industry practices. As pharmaceutical markets in genetic drugs continue to grow, pulling resources across academia and industry has all led to the questions of scalability and thus profitability, which the NS protocol has demonstrated to answer.