IEX Purification of RNA Base(s) Containing DMT-on Oligonucleotide Single Strand Using a One Step On-Column Detritylation Technique

Abstract

The purity of a drug product is an important aspect for successful drug development to maximize drug efficacy and reduce potential toxicity arising from impurities. In the case of oligonucleotide synthesis, shorter oligomers may be produced along with final full-length oligonucleotides upon completion of the synthesis process. This is potentially due to incomplete reactions and reagents used in the synthesis process. It is important to design strategies to optimize drug product quality through implementation of appropriate synthesis and purification steps. Automated solid phase oligonucleotide synthesis is a commonly used approach to generate small and large-scale single strand material and has been established as a robust method for synthesizing oligonucleotide at various scales. In this process, to ensure correct chain growth, acid labile Di-methoxy-trityl (DMT) group is utilized to protect the 5’ Hydroxyl (OH) group of the ribose sugar in the growing oligonucleotide chain. The 5’ hydroxyl group is kept on the incoming phosphoramidite and removed prior from the nucleoside present on the solid support to grow the sequence in the 3’-5’ direction. Oligonucleotide sequences can be synthesized either with the 5’ Di-methoxy-trityl (DMT) group remaining “on” or “off” at the 5’ end position for the final base. This is done by avoiding the final acid treatment (detritylation) to remove the DMT group of the last base added at the end of synthesis cycle. Presence of the DMT group on the last base of oligonucleotides, increases the hydrophobicity significantly and provides a great chromatographic handle to aid hydrophobicity-based chromatography. Here, we propose to evaluate a purification procedure for oligonucleotides by incorporating the DMT at the 5’ end, such that it can act as an anchor and provide strong binding to the chromatographic stationary phase used in IEX chromatography. In general, Purification of DMT-on oligonucleotides requires a multistep, time consuming process to remove undesirable impurities. We anticipate that DMT-on purification on IEX stationary phases will allow for a direct on column detritylation step as part of the purification process, hence eliminating the need of additional post purification steps, which are difficult to scale and with the potential to improve the product purity without compromising yield.