Manufacture of therapeutic oligonucleotides: Development of new reagents and processes

The solid phase synthesis of oligonucleotides at smaller scales is well understood and the processes at this scale do not impose any impact on economic, environmental or safety considerations. However, apart from economic consideration, significant challenges still exist for large-scale manufacture. More than a decade’s effort and continuing searches for therapeutic agents based on antisense, ribozyme, immunostimulatory, aptamer, decoy and recent RNAi technologies have provided impetus for the largescale manufacture of therapeutic grade oligonucleotides. Currently there are more than 200 oligonucleotides under preclinical and clinical investigations for various diseases like cancers, autoimmune, asthma, and allergy. The clinical investigations require synthetic olignucleotides ranging from a few-grams to several kilograms. The synthetic oligonucleotides under investigations are classified as phosphorothioate oligonucleotides (first generation modified), chimeric oligonucleotides (second generation modified oligonucleotides where sugar and/or heterocyclic base and internucleotidic linkages are modified), short dsDNA (double stranded deoxy nucleic acids), dsRNA (double stranded ribonucleic acids) as well as standard phosphate diesters for aptamer technology. The structure of these various oligonucleotides are presented in Figure 1. Oligonucleotide syntheses, irrespective of the scale or type (DNA, RNA, aptamer etc.) are performed using β-cyanoethyl-phosphoramidite chemistry using solid support in an automated synthesizer. The synthesis proceeds from 3′-end to 5′-end of the sequence. The 3′-end nucleoside is covalently attached to the solid support in such manner that, at the end of synthesis, the covalent linkage can be removed easily. The chemistry utilized and steps involved in the synthesis of oligonucleotides are represented in the Scheme I and Figures 2, 3 and 4. The chemical reactions in solid support mediated synthesis are almost quantitative, driven by the use of excess reagents. In smaller scales of synthesis, the use of excess amount of reagents, chemicals and solvent may not have significant impact on costs, environmental safety and availability of raw materials supply. However, at larger scales (100 to 500 mmole) all of these issues become major hurdles for the development of oligonucleotide as a viable drug product. The challenges for large-scale oligonucleotide synthesis can be outlined as: