A luminogenic real-time detection system for monitoring enzymatic manufacturing of oligonucleotides – LuminoManufacOligo

In drug discovery, new modalities are chemical entities (e.g., peptides, oligonucleotides, carbohydrates, …) of intermediate size between small molecule therapeutics and biologics (i.e., proteins, antibodies). They aim at combining the efficient cell intake of small molecules with biologics' ability to interact with large targets like protein-protein interations. Among these new modalities, oligonucleotides have been investigated during the last 20 years and with currently more than 135 candidates in clinical trials the medical community expects a wave of oligonucleotide therapeutics reaching the market in the near future. The excitement for these drugs addressing unmet medical needs also raises challenges regarding their manufacturing in large quantities. Oligonucleotide manufacturing is currently performed on solid-phase using the phosphoramidite chemistry and because of the large excess of reagents/solvents and chromatography purifications it is not an environmentally and economically sustainable process. An alternative strategy currently under investigation in the industry is a biocatalyzed synthesis using polymerase enzymes to link nucleotides to each other. In order to ensure the successful completion of the enzymatic step an analytical method reporting in real-time the advancement of the reaction would be very useful in the manufacturing plant. In this context, the goal of our project is to develop a "Process Analytical Technology" (PAT) tool for the detection of the pyrophosphate ion (PPi) released during the polymerase-catalyzed reaction. PAT tools are very sought after in the manufacturing plant because of the benefits they provide in terms of cost/time savings as well as process safety. Their availability can drive the choice of a process route over another and we believe it is an important aspect of the advanced manufacturing technologies necessary to lead to sustainable processes and industrial innovation/renewal. As we anticipate selectivity issues for differentiating between the detection of PPi over the dNTPs used in the enzymatic reaction we suggest to break down PPi into more reactive phosphate ions (Pi) and propose three types of reaction-based small molecule luminescent probes to detect/quantify it. The first approach would be to synthesize a conventional fluorescent chemodosimeter from an aniline/phenol-based fluorophore equipped with a suitable Pi-sensitive triggering unit. Fluorescence is restored only after deprotection of the fluorogenic center of the probe by Pi (fluorogenic "OFF-ON" detection). Because in practice it is often difficult to completely switch "OFF" the fluorescence of an aniline/phenol-based fluorophore solely through masking of its amino/hydroxyl group, the second approach we propose is to build in situ the push-pull conjugated backbone of the fluorophore from a "caged" precursor via a cascade reaction triggered by Pi. This novel probe design known as "covalent assembly" principle provides reaction-based fluorescent probes with zero background signal, particularly useful for the present application requiring high detection sensitivity. As a third approach, we propose the development of chemiluminescent probes where the excitated state of the emitting species is reached thanks to a chemical reaction instead of external excitation. The absence of excitation removes all background noise from (bio)molecules (and possible damages of them) in the reaction medium and should result in a further increase in sensitivity. We will do the evaluation of the three types of probes in the presence of Pi and in the context of the enzymatic synthesis of oligonucleotides and identify the optimum candidate(s) for further use. Our ultimate objective will be to provide an analytical tool ready to be translated in the manufacturing plant and potentially be useful to other biotechnology applications requiring the monitoring of polymerase activities (qPCR, DNA sequencing, …).