Next-Generation Synthetic and Biocatalytic Strategies for Therapeutic Oligonucleotide Production: A Process Chemistry Perspective

The rapid expansion of oligonucleotide therapeutics has created unprecedented demand for robust, scalable, and economically sustainable manufacturing technologies. While solid-phase phosphoramidite synthesis remains the industry standard for manufacturing antisense oligonucleotides, siRNAs, guide RNAs, aptamers, and other modified oligonucleotides, increasing sequence complexity, extensive chemical modifications, ligand conjugation, sustainability concerns, and manufacturing costs continue to challenge conventional production processes. This review provides a comprehensive process chemistry perspective on next-generation oligonucleotide manufacturing by integrating advances across chemical synthesis, raw material supply, bioconjugation, and emerging biocatalytic technologies. We first review the evolution of phosphoramidite chemistry, including the industrial manufacture of nucleoside phosphoramidites, controlled pore glass and polystyrene supports, synthesis reagents, and modified nucleotide building blocks that underpin commercial oligonucleotide production. The discussion then highlights downstream process development, purification strategies, and manufacturing considerations associated with large-scale oligonucleotide production, followed by recent advances in site-specific bioconjugation with ligands including GalNAc, lipids, peptides, and antibodies that have significantly expanded the extrahepatic delivery of therapeutic oligonucleotide drugs. We emphasize on emerging enzymatic and chemoenzymatic manufacturing platforms, including early ligase-mediated fragment assembly, template-independent terminal deoxynucleotidyl transferase-based controlled enzymatic synthesis, the industrial translation represented by Codexis ECO Synthesis, and the EnPlusOne ezRNA protocols. Finally, we discuss the need for expanding supply chain for modified nucleoside triphosphates, engineered polymerases, ligases, and accessory enzymes. Collectively, these advances demonstrate how the convergence of synthetic chemistry, enzyme engineering, and industrial bioprocess development is reshaping oligonucleotide manufacturing and establishing the foundation for next-generation, cost-effective, and environmentally sustainable production of increasingly complex nucleic acid therapeutics.