(567f) Liquid Phase Membrane Supported Cyclical Flow Synthesis of Oligonucleotides for Therapeutic Applications

There now exists a consensus that RNA based gene inference techniques such as Anti-Sense Oligonucleotides and siRNA represent a significant therapeutic modality in the pharmaceutical industry. While the applications and understanding of the underlying biology of these systems for use as therapeutic platforms have advanced rapidly, little change in the commercial synthesis of these compounds has occurred. Metric tonnes per annum of oligonucleotides are projected to be required due to the high number of oligonucleotide-containing drugs in late phases of clinical trials and also projected for individual ASO therapies as larger patient populations are targeted in clinical trials following a number of clinical successes in small orphan patient populations.^[1][2] The existing state-of-the-art synthesis of oligonucleotides is carried out with the use of a solid phase support and batch sizes currently limited to approximately 5 kg due to the constraints of solid phase synthesis.^[3] In contrast, liquid phase supports reduce many of the limitations that are associated with solid phase supports.^[4] Liquid phase synthesis of oligonucleotides was established by using linear and branched polyethylene glycol (PEG) as the soluble support^[5] Membrane enhanced synthesis of oligonucleotides and peptides using PEG as a soluble support have been shown to be a potential solution for the problems associated with solid phase synthesis.^[6][7]

The current work demonstrates proof of concept for a novel liquid phase manufacturing process utilizing cyclical flow synthesis combined with a novel membrane separation (Figure 1). This should enable fully scalable cyclical flow synthesis of oligonucleotides with increased productivity within a compact and scalable reactor footprint. This novel process intends to meet the growing demands of the industry and promises reduced capital costs and increased product quality control.

Modifications to soluble supports were utilized to both optimize the nucleotide loading process but also to optimize membrane flux and rejection factor, and hence yield and purity with commercially available membranes. Both liquid batch and phase cyclical flow synthesis of short oligonucleotides were optimized for coupling yield, phosphoramidite consumption, solvent consumption and overall productivity. The ability to use optimum reaction/separation timescales which are inherent to coupling reactions in oligonucleotide synthesis enabled improved system performance and in turn the ability to rapidly supply oligonucleotides on demand.

Acknowledgements:

This publication is supported by Science Foundation Ireland (SFI) through SSPC, The SFI Research Centre for Pharmaceuticals (Grant number 12/RC/2275_P2).

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