The global market for RNA therapeutics is projected to reach $25 billion by the early 2030s, a clear signal of the modality’s move into mainstream medicine. No longer confined to rare genetic disorders, these highly specific, gene-targeted treatments have now been approved for widespread chronic conditions like cardiovascular disease. This shift represents a massive change in production demands, forcing the industry to redefine the manufacturing capabilities needed for the synthesis of RNA oligonucleotides.
New Possibilities and Escalating Demand
A combination of clinical success and an expanding range of approved indications drive the momentum behind RNA therapies.
Unlike earlier RNA-based treatments designed for smaller patient populations, a growing number of recently approved therapies, as well as a high percentage of assets currently in pre-clinical and clinical stage are being developed for chronic administration to millions of patients globally. This trend is exemplified by assets targeting cardiovascular disease, such as Leqvio® (inclisiran), a cholesterol-lowering drug with an estimated patient population in the tens of millions globally, as well as lepodisiran and olpasiran, currently in phase III clinical trials, for lowering lipoprotein(a) in a projected 60+ million patients in the US. To meet market demand, estimates anticipate production of therapeutic RNA oligonucleotides to more than quadruple to 30+ metric tons of API by the end of this decade. These predictions underscore a critical need: manufacturing approaches that can reliably deliver therapeutic RNA oligonucleotides at scale for life-saving therapies.
The Legacy Challenge: Why Traditional Chemistry Can't Keep Up
For decades, solid-phase oligonucleotide synthesis (SPOS) has served as the foundation of RNA production. It enables the stepwise assembly of oligonucleotides with precise sequence control and remains a reliable method for early-stage development and for shorter constructs. However, as therapeutic RNA advances in both scale and sophistication, the limitations of this approach have become increasingly apparent.
A Cycle of Compromise: Yield Loss and Impurities
SPOS relies on a repetitive four-step chemical cycle, coupling, capping, oxidation, and detritylation, performed on a solid support. While individual steps are efficient, the overall yield decreases exponentially with sequence length, which presents a challenge, particularly for longer RNA constructs. Incomplete reaction cycles and repeated exposure of the growing oligonucleotide chain to harsh chemical conditions result in truncated strands and a multitude of other impurities, consequently necessitating extensive and costly purification steps. This cumulative yield loss becomes a significant barrier to efficient, large-scale manufacturing of oligonucleotides.
The Cleavage Conundrum: A Delicate Final Step
The final steps in SPOS, cleavage and deprotection, add to the challenges of RNA impurities due to the inherent chemical lability of oligonucleotides. During the removal of the final oligo product from its solid support, standard cleavage agents can induce strand scission, especially at elevated temperatures. Subsequent chemical deprotection must be carefully sequenced to avoid over-reaction, resulting in product degradation, or under-reaction, causing incomplete removal of protecting groups. Each new sequence usually requires extensive re-optimisation of these conditions, adding significant time and cost to the development process.
The Burden of Scale: Solvents and Infrastructure
SPOS is a solvent-intensive process, requiring large volumes of flammable and hazardous chemicals like acetonitrile, toluene, and dichloromethane. Facilities producing multi-kilogram quantities must invest heavily in specialised infrastructure, including solvent tank farms and explosion-proof systems, which significantly increases capital and operational expenditure. For example, a major life sciences company recently announced an investment of nearly three-quarters of a billion dollars to expand its manufacturing capacity by one metric ton annually using these traditional chemical methods. This illustrates the substantial financial commitment required, especially in light of the anticipated rapid growth in production demand mentioned above.
A Mismatch with Modern Goals
The environmental footprint of SPOS is increasingly misaligned with industry environmental, social, and governance (ESG) goals. This solvent-heavy workflow generates considerable chemical waste and emissions, while also raising concerns around worker safety and regulatory compliance. As sustainability becomes a board-level priority, traditional synthesis methods face mounting pressure to adapt to cleaner, more efficient practices.
A New Blueprint for Production: The Promise of Enzyme-Enabled Synthesis
While SPOS remains a workhorse for many applications and is still a robust and versatile method for handling small-scale oligonucleotide synthesis, the high-scale production of RNA therapeutics has created a new, unmet need. In response to these operational limitations, enzyme-enabled RNA synthesis is emerging as a powerful, forward-compatible alternative for large-scale manufacturing of therapeutic oligonucleotides.
Unlike traditional chemical methods, enzymatic workflows operate under mild, fully aqueous conditions, hence eliminating the need for protecting groups and hazardous solvents. These approaches typically use engineered polymerases to sequentially add monomers to generate a full-length strand or, in combination with ligases, to assemble full-length oligonucleotides from shorter, high-purity fragments. Critically important, the use of enzymes as (bio) catalysts makes this manufacturing solution inherently more scalable by allowing the process to operate with the growing oligonucleotide chain in solution while retaining the enzyme immobilised on a column. Besides improving scalability, keeping the oligonucleotide in solution also offers greater process control by allowing real-time, in-line monitoring of reaction progress via advanced analytical methods such as high-performance liquid chromatography and mass spectrometry. For these reasons, this strategy is particularly well-suited to meet the high-volume demands of tomorrow’s therapeutic RNA assets. Beyond simpler, efficient and more sustainable oligo synthesis, enzymatic approaches can streamline downstream processing by reducing purification demands, providing key advantages for developers navigating compressed timelines and growing demand for large-indication APIs.
Ligation: The Advantages of a Modular Approach
One of the most exciting applications of enzyme-enabled oligonucleotide synthesis lies in the highly versatile, ligation-based assembly of full-length RNA therapeutics. This is a fundamental shift from the traditional linear, step-by-step synthesis using SPOS. Instead of building RNA strands from scratch on a solid support, enzymatic workflows can leverage the spontaneous self-assembly of short, complementary oligonucleotide fragments into the characteristic duplex structure of nucleic acids, followed by efficient joining of adjacent fragments with the help of ligases. This modular strategy offers several distinct advantages.
Optimised Yield and Quality
Short RNA fragments, which can be synthesised either enzymatically or chemically, require fewer iterative cycles during production, thereby reducing the cumulative yield losses and impurities. In turn, improved yields and quality of fragments can simplify downstream processing and, in combination with process control strategies to suppress incorporation of impurities during ligation, directly translate into higher overall efficiency for production of full-length therapeutic oligonucleotides.
From Wild-Type to Engineered Enzymes
The success of ligation-based workflows is highly dependent on the performance of the ligase itself. Naturally occurring, or wild-type (WT) ligases, pose significant limitations for commercial manufacturing. They often struggle with chemically modified RNA inputs, display poor activity at high substrate concentrations, and are incompatible with preferred process conditions.
To overcome these barriers, protein engineering has been critical. Engineered ligases are specifically re-tooled to tolerate chemical modifications typically found in RNA therapeutics and to exhibit significantly enhanced catalytic activity and robust performance under process conditions. These performance characteristics maximise ligation efficiency while minimising enzyme loadings, resulting in exquisite product quality and substantial economic upside.
Applying Enzyme-based Manufacturing Solutions
Enzymatic manufacturing offers versatility and adaptability in oligonucleotide synthesis today. Beyond ligase-based processes, already scaled to multi-kilogram production and supported by a recent $250 million infrastructure investment from a major drug innovator, development is advancing rapidly in sequential synthesis of oligonucleotide fragments and full-length RNA therapeutics through stepwise enzymatic polymerisation of modified nucleotides. Over the past three years, breakthroughs in enzyme engineering and process development have moved this approach from benchtop experiments to pilot-scale operations. It now achieves coupling efficiencies comparable to SPOS, continues to scale steadily, and shows strong potential to surpass current methods in product quality. Together with ligase-based assembly, these innovations are paving the way toward fully enzymatic RNA oligonucleotide production, reshaping the future of RNA therapeutics.
Delivering on the Promise: A Scalable and Sustainable Future for RNA Medicine
The diversification of structure and broader applications of RNA therapeutics demand new manufacturing solutions. With drug development expanding beyond the rare disease space to indications affecting tens of millions of patients, innovation must not remain confined to discovery but extend to novel processes addressing today’s scalability and sustainability challenges. Only this way can we ensure that the extraordinary promise of these molecules translates into meaningful clinical impact and improved patient outcomes.
Enzyme-enabled processes represent a transformative innovation, offering a powerful alternative that reflects the changing needs of the field. The scalable, efficient, and sustainable production of RNA oligonucleotides is uniquely enabled by the versatility of the inherently adaptable toolbox of engineered enzymes. Integrated into modern manufacturing platforms, these biocatalysts deliver on the expectations for quality, speed, compliance, and environmental performance, critical factors that will accelerate the realisation of RNA therapeutics’ full potential.
References
1. Allied Market Research. (April 2025). RNA Based Therapeutics Market by Type, Application, and End User: Global Opportunity Analysis and Industry Forecast, 2021–2030. https://www.alliedmarketresearch.com/RNA-based-therapeutics-market
2. Thanassoulis, G. (2019). Screening for High Lipoprotein(a). Circulation, 139(12), 1493–1496. https://doi.org/10.1161/circulationaha.119.038989
3. Tsimikas S. Lipoprotein(a) in the year 2024: A look back and a look ahead. Arterioscler Thromb Vasc Biol. 2024;44(7):1485–1490. https://doi.org/10.1161/ATVBAHA.124.319483
4. Example scenario based on Leqvio dosing of 284 mg per administration with twice-yearly maintenance dosing. Per asset per year: 10,000,000 patients × 284 mg × 2 = 5,680 kg. For five assets: 28,400 kg or 28.4 metric tons per year. Patient assumption from HSBC Global Investment Research Dec 2025. Dosing source: Leqvio HCP, Dosing and Administration, https://www.leqviohcp.com/dosing-and-administration; Patient population assumption source: HSBC Global Investment Research, 2026 in Pharma catalysts: Reflecting on lessons from 2025, December 2025.
5. Glen Report 20.24: Deprotection – Volume 1 – Deprotect to Completion. https://www.glenresearch.com/reports/gr20-24
6. Glen Report 21.15: Deprotection – Volume 2 – RNA Deprotection. https://www.glenresearch.com/reports/gr21-15
7. Agilent Technologies Inc. (January 10, 2023). Agilent Investing $725 Million to Expand State-of-the-Art Manufacturing Capacity for Production of Nucleic Acid-Based Therapeutics. Press Release. https://www.agilent.com/about/newsroom/presrel/2023/09jan-gp23002.html.
8. Alnylam to Invest $250 Million to Add Enzymatic Ligation Platform to U.S. Manufacturing Facility to Meet Growing Global Demand for RNAi Therapeutics. (n.d.). Investor Relations | Alnylam Pharmaceuticals, Inc. https://investors.alnylam.com/press-release?id=29466
