In the last decade, RNA therapeutics have surged from early scientific promises to being an established part of the modern drug development landscape. Today, modalities such as small interference (si)RNA, antisense oligonucleotides (ASOs), and single guide (sg)RNA offer novel, potent and precise treatment options across a growing spectrum of diseases, benefiting patients with both rare and common conditions. To meet growing demand for therapeutic oligonucleotide synthesis, innovative new manufacturing solutions are needed to overcome scale, capacity, and quality limitations of existing chemical solid-phase oligonucleotide synthesis (SPOS) technology.
The global RNA therapeutics market is projected to reach $25 billion by 2030. The five-fold expansion since 2021 can be attributed to increasing clinical demand, driven in equal parts by a broader spectrum of disease targets and a shift from low-volume, rare disease space to chronic indications with patient populations in the millions. The transition from early RNA-based treatments serving small patient populations to high-volume assets is exemplified by Leqvio (inclisiran), a cholesterol-lowering drug with an estimated eligible patient population of over 20 million in the US alone. Similarly, programs like lepodisiran and olpasiran, both in phase III development at the time of writing, for lowering lipoprotein(a) in a projected 60+ million patients in the US showcases the modality’s expansion into large-population, chronic indications. These examples also underscore the need for new manufacturing solutions, requiring robust, reliable, and scalable approaches to deliver hundreds to thousands of kilograms of API.
The SPOS limitation
Chemical RNA synthesis by SPOS has played – and will continue to play – a central role in advancing RNA therapeutics. However, the technology faces significant challenges when it comes to efficient large-scale manufacturing of RNA-based medicines.
Small batch sizes: SPOS is typically capped at batch sizes of 5 to 10 kilograms due to physical constraints resulting from oligonucleotide immobilization on solid support. For high-volume APIs, production can easily require tens to hundreds of individual batches, posing major logistics and quality assurance challenges.
Compromised quality: The quality of RNA therapeutics is impacted by repeated exposure to harsh chemical conditions during iterative SPOS cycles and during the cleavage and deprotection steps at the end of the synthesis. Mitigation strategies such as removal of impurities by chromatography in downstream processing compromise product yields and add operational expenditure.
Operational burden at scale. SPOS is highly solvent-intensive, requiring large volumes of flammable, hazardous chemicals such as acetonitrile, dichloromethane and toluene. Facilities producing multi-kilogram quantities of RNA must invest in solvent tank farms, explosion-proof systems and specialized waste management infrastructure – significantly increasing capital and operational expenditure.
Sustainability and ESG impact. The environmental footprint of SPOS is increasingly misaligned with industry environmental, social, and governance (ESG) goals. Solvent-heavy workflows generate chemical waste and emissions, while raising concerns around worker safety and regulatory compliance. As sustainability becomes a board-level priority, traditional synthesis methods face mounting pressure to adapt.
While specific limitations of SPOS technology are being addressed through innovations such as liquid-phase synthesis, novel reactor architecture, solvent recycling, and continuous purification, these efforts are largely incremental and often introduce new challenges even as they solve existing ones.
Reenvisioning RNA manufacturing with enzymes
Enzyme-enabled oligonucleotide synthesis is widely regarded as the alternative and logical next chapter in industrial scale production of RNA therapeutics. By merging nature’s exquisite ability for nucleic acid synthesis and AI/machine learning based technologies in protein and process engineering, enzymatic workflows offer a clear path towards greater scalability and improved product quality and yields while largely eliminating hazardous solvents and reducing process complexity.
These approaches typically rely on polymerases and ligases to assemble full-length oligonucleotides, starting from individual nucleotides and fragments, respectively. While leveraging these enzymes’ inherent catalytic abilities, their functional characteristics can be tuned by protein engineering to turn them into biocatalysts with reliable performance under manufacturing conditions for therapeutic-grade material. Biocatalysts used in large-scale RNA synthesis are built for stability and activity to maximize space-time yield, as well as for tolerance towards chemically modified nucleotides (e.g. 2’-OMe, 2’-F, phosphorothioates) which constitute the majority of building blocks in today’s siRNA therapeutics and are increasingly integral to tomorrow’s RNA medicines.
Upon incorporation into manufacturing processes, these engineered enzymes allow for oligonucleotides to stay in solution, eliminating the scalability constraints of SPOS. They operate under mild, aqueous-phase conditions which remove the need for hazardous solvents and reduce by-products, hence improving product quality. In turn, the gains in quality can simplify downstream processing by reducing or even eliminating purification demands, benefiting yields, and compressing timelines.
RNA manufacturing with enzymes is not science fiction! Already today, ligases are effectively utilized in manufacturing RNA therapeutics for clinical applications at multi-kilogram scale. The approach represents a natural bridge between traditional chemical synthesis and fully enzymatic processes, offering several key advantages:
Modularity. Ligases allow for modular construction of complex RNA sequences, enabling the joining of short RNA fragments into full-length therapeutic oligonucleotides. RNA fragments can be synthesized by chemical or enzymatic approaches, adding flexibility in supply chain and versatility for integration of other enzymatic tools in the manufacturing process.
Technical advantages. Beyond the benefits of scalability and sustainability highlighted above, the use of short RNA fragments avoids the cumulative quality and yield losses associated with long, stepwise SPOS. It enables developers to more effectively manage sequence complexity, structural modifications, and simplify purification workflows.
Commercial and regulatory momentum. The use of ligases in clinical-scale RNA manufacturing lends credibility and regulatory familiarity to enzymatic methods. This can accelerate adoption across the industry.
At the same time, the development of manufacturing solutions for the fully enzymatic synthesis of RNA fragments and full-length oligonucleotides is advancing rapidly. Already, highly engineered polymerases have demonstrated successful polymerization of chemically modified nucleotides into therapeutically relevant sequences at scales reaching tens of grams. Notably, they have done so by readily matching the coupling efficiency of SPOS while eliminating hazardous solvents and increasing product yields – a result of fewer side products and simpler purification needs.
These efforts have also demonstrated the process’ capability to synthesize oligonucleotides directly in solution - a key advancement over traditional SPOS. This breakthrough introduces a novel and elegant approach to effectively overcome the significant scalability limitations of current oligonucleotide synthesis technology. In addition, it enhances manufacturing control by enabling real-time reaction monitoring through in-line analytics. At its current pace of development, this first-generation fully enzymatic oligonucleotide manufacturing platform is poised to deliver kilogram-scale quantities of GMP-grade RNA therapeutics in the next 18-14 months.
Innovation in oligonucleotide synthesis continues. As the pipeline for RNA therapeutics expands and expectations around speed, compliance, and environmental performance intensifies, the ability to adapt processes around these molecules will define the next generation of RNA production. Given their developmental trajectory, enzymatic workflows are poised to deliver on these expectations, promising a robust yet versatile foundation for the future of RNA oligonucleotide manufacturing.
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