Oligonucleotide therapeutics (oligos) treat disease at the RNA-level, enabling targets to be reached beyond those generally accessible to conventional pharmaceuticals. Since the Nobel Prize winning discovery of small interfering RNA (siRNA), its specificity and selectivity have driven the pharmaceutical industry to design 15- to 25-mer oligonucleotide analogues as a new class of drugs. Already, 11 oligo drugs are approved for use in humans, with over 150 in clinical trials.
Initially, oligo therapeutics targeted rare diseases, but in recent years an increasing number have shown excellent results for diseases with large patient populations. For instance, Inclisiran, developed for the treatment of cardiovascular disease where there is a need for tons-per-annum, was approved for use in the NHS in 2021. However, the high cost and limited scalability of manufacturing these complex molecules using classical solid phase synthesis (SPS) is holding back these benefits. These shortcomings have motivated us to seek an improved technology for oligo manufacture.
Current oligo production uses stepwise, or iterative SPS with P(III) phosphoramidite chemistry to assemble defined sequences of nucleotide monomer building blocks (BBs) into oligo drugs. After each chain extension reaction, the solid support beads are washed to remove debris and excess reagents, ready for another cycle. However, the washing of packed beds of support in ever larger diameter columns with thicker bed depths is very challenging to scale up. Furthermore, although P(III) chemistry is fast and has high coupling efficiency, the monomers are expensive and sensitive. Therefore, new separation methods and alternatives to P(III) chemistry are sought by the pharmaceutical industry.
Nanoligo addressed both alternatives to P(III) chemistry and to SPS in three ways: Purification of growing oligos was effected in solution phase using scalable membrane modules; new BBs that maximise the solubility of growing oligos, and minimise the viscosity of the synthesis solution, greatly facilitating solution phase oligo synthesis were tested Nanostar Synthesisers; cheaper, more stable H-phosphonate BBs were prepared, and used to make oligos in a Nanostar Synthesiser.
The physical roadblock of packed support beds can be avoided by performing all the chemistry in solution phase, which can be scaled up almost indefinitely. However, a method must then be found to remove reaction debris after every cycle of chain extension. For this purpose, we are now developing solution phase oligo synthesisers built around size-selective membrane separation. In principle, this is highly scalable because the chemistry industry knows how to circulate and pressurise large volumes of liquid. Before Nanoligo, we had only proved the concept using coffee cup-sized sheets of membrane clamped in flat cells with classical phosphoramidite building blocks (BBs). The process was very slow due to the small membrane area and impossible to scale. It was essential to change to a compact, scalable membrane format. Nanoligo has enabled this, firstly through the consistent production of large areas (many square metres) of PBI nanofiltration membrane - scaling up from square centimetres is non-trivial! Secondly, the PBI membrane was incorporated into Spiral Wound Modules (SWMs), a technology developed for compressing large areas of membrane into a small volume. This approach has been known for decades in the water purification industry, but could not have been applied to our unique polymer membranes developed for organic solvent nanofiltration without the Nanoligo project.
