RNA-based therapies have rapidly emerged as a breakthrough in medicine, holding considerable promise for tackling a wide range of diseases, from rare genetic disorders to prevalent chronic conditions such as cardiovascular disease. These innovative treatments harness the power of RNA to target and modify cellular processes with precision, offering a level of control previously unimaginable. Their potential is reflected in the growing RNA therapeutics market, projected to increase in value to $18 billion by 2030, expanding at a robust compound annual growth rate of 5.6% (1). What was once a relatively niche field is now addressing broader, higher-volume indications, with treatments such as inclisiran (LEQVIO) for atherosclerotic cardiovascular disease, fundamentally redefining expectations for drug development.
However, this explosive growth and the shift toward large-patient-population applications have cast a spotlight on a critical bottleneck: manufacturing. The ability to deliver high-purity RNA constructs at scale — quickly, cost-effectively and sustainably — has become a defining challenge for both therapeutic developers and manufacturers. For decades, solid-phase oligonucleotide synthesis (SPOS) has been the foundation of RNA manufacturing (2). While reliable, its dependence on solid-phase workflows, repeated chemical cycles and large volumes of hazardous solvents presents significant practical, economic and, crucially, environmental limitations.
The unsustainable legacy of SPOS
SPOS is a stepwise process where RNA sequences are built one nucleotide at a time through a series of chemical coupling reactions on a solid support. Its reliability and precision have made it the industry standard for constructing a wide range of RNA sequences, particularly for early-stage development and clinical trials. However, as RNA therapeutics transition from rare disease applications to large-volume, chronic indications, the inherent limitations of SPOS have become increasingly apparent, exposing significant environmental and operational challenges.
One of the most pressing concerns is the high consumption of solvents. SPOS processes rely heavily on large volumes of flammable and hazardous organic solvents such as acetonitrile, dichloromethane and toluene. These solvents are crucial for various steps, including deprotection, coupling, capping and oxidation. The sheer quantity of solvent used generates a staggering amount of chemical waste; for example, traditional methods can result in a Process Mass Intensity (PMI) of approximately 4,300 kg of waste per kilogram of active pharmaceutical ingredient (API) for a typical 20-building block oligonucleotide (3). Managing and disposing of this hazardous waste incurs substantial costs and poses significant environmental risks.
Beyond solvents, SPOS involves the use of harsh chemical reagents and protecting groups. These chemicals, while necessary for precise nucleotide assembly, can introduce undesirable side reactions and generate structurally diverse impurities. Among these are Class IV impurities, which are notoriously challenging, if not impossible, to remove through conventional purification methodologies. The presence of such impurities requires extensive and often energy-intensive downstream purification steps, further exacerbating the environmental footprint through increased solvent use and energy consumption.
The operational burden of SPOS at scale is also considerable. Facilities producing multi-kilogram quantities must invest in specialized infrastructure, including solvent tank farms and explosion-proof systems, to safely handle and store these hazardous materials. This translates into capital-intensive scale-up, exemplified by Agilent’s $725 million investment to expand capacity for traditional RNA manufacturing (4). Ultimately, the reliance of SPOS on hazardous chemicals, high waste generation and significant infrastructure demands increasingly clash with evolving sustainability and environmental, social and governance (ESG) goals, pushing the industry to seek greener, more responsible manufacturing alternatives.
Scalable and efficient RNA therapeutics
Enzymatic RNA synthesis offers significant improvements in scalability and efficiency, which are crucial for meeting the growing global demand for RNA therapeutics. Traditional SPOS methods face intrinsic scale limitations, with yields typically peaking at over 5 kg per synthesis run. This constraint becomes a significant bottleneck as RNA therapies target larger patient populations, demanding multi-metric ton production capabilities. Enzymatic synthesis, by contrast, has demonstrated the ability to scale into double digits (of kilograms) per run, significantly exceeding the output of many optimized SPOS workflows.
This superior scalability is attributed mainly to the robust performance of engineered enzymes, which maintain high activity even at elevated substrate concentrations. This characteristic, coupled with simplified reaction conditions and modular process design, enables more predictable and efficient scale-up, facilitating seamless technology transfer into Good Manufacturing Practice (GMP) environments. For instance, the solution-phase nature of sequential enzymatic synthesis enables larger reaction volumes and more efficient mixing, overcoming the mass transfer limitations often encountered in solid-phase chemistry. Similarly, the modularity of ligation-based assembly is a key enabler for high-volume production. By allowing for the parallel synthesis of shorter, high-purity fragments, this approach significantly increases overall throughput. It accelerates the manufacturing cycle, making it particularly well-suited for the rapid production of therapeutics for chronic, population-wide use.
Another critical advantage lies in enhanced purity and quality. Enzymatic workflows, whether sequential or ligation-based, inherently reduce the formation of undesirable byproducts compared to chemical methods. This is particularly true for problematic Class IV impurities, which are minimized or eliminated, resulting in a higher-quality therapeutic-grade RNA product. In ligation-based workflows, the ability to start with highly purified RNA fragments, combined with the selectivity of engineered ligases, results in cleaner final assemblies. Higher purity ensures product safety and efficacy, directly translating into reduced rework and less material waste during subsequent purification steps, which further contributes to the overall sustainability of the process.
These improvements in purity and scalability directly contribute to streamlined production timelines (5). Enzymatic synthesis can significantly reduce the overall time from raw material to final product (5, 6). While individual cycle times for sequential enzymatic synthesis may currently be comparable to or slightly longer than those for SPOS, the ability to run much larger batches and achieve higher material outputs per run fundamentally offsets this difference (5, 7). For ligation-based approaches, the parallel synthesis of fragments enables flexible, modular process design, supporting faster manufacturing cycles and quicker responses to market demand (8).
A greener choice for RNA manufacturing
The environmental footprint of traditional phosphoramidite chemistry has prompted a focused shift toward enzymatic RNA synthesis as a transformative, next-generation solution (5, 9). This innovative approach represents a fundamental change, leveraging the power of engineered enzymes to construct oligonucleotides under mild, aqueous conditions (5, 9). This move away from harsh organic solvents and protecting groups is the cornerstone of its sustainability advantages, offering a fundamentally different and significantly greener pathway for RNA production (5).
Unlike SPOS, which requires large volumes of flammable and toxic organic solvents, enzymatic workflows mimic natural biological processes, which occur predominantly in water. This eliminates the need for many hazardous chemicals, drastically reducing the generation of chemical waste and the associated complexities of safe handling and disposal. This aqueous environment enhances the safety profile for manufacturing personnel and streamlines facility requirements, eliminating the need for explosion-proof systems and extensive solvent recovery infrastructure.
Enzymatic synthesis platforms typically employ two distinct approaches, each offering unique sustainable advantages:
Firstly, fully sequential enzymatic synthesis employs a cyclic process to construct RNA strands one nucleotide at a time. In this workflow, engineered enzymes, often immobilized on solid supports, facilitate the precise addition of nucleotides in a solution-phase environment. The process typically involves an extension step, where a packed-bed reactor containing immobilized polymerase enables the controlled, directional extension of the RNA sequence. This is followed by a deblocking step, utilizing an immobilized phosphatase to remove blocking groups and prepare the strand for the next cycle. Compared to SPOS’ four-step chemical cycle, this streamlined, two-step enzymatic cycle (extension and deblocking) significantly reduces the number of chemical reactions and associated byproducts. The mild, aqueous conditions further minimize the formation of undesirable impurities, contributing directly to a cleaner synthesis process and less waste. This modular and “green” approach is particularly well-suited for RNA sequences with modified nucleotides, all while maintaining an environmentally benign operational profile.
Secondly, for more complex or longer RNA constructs, ligation-based assembly offers a highly efficient and sustainable alternative. This modular strategy involves synthesizing shorter, high-purity single-stranded RNA (ssRNA) fragments, either enzymatically or through traditional SPOS, and then joining them together using engineered RNA ligases designed to assemble double-stranded constructs. This approach provides several key sustainable benefits. By assembling pre-purified, shorter fragments, it effectively circumvents the cumulative yield losses inherent in stepwise SPOS, which can lead to significant material waste for more extended sequences. This modularity allows for the parallel synthesis and purification of fragments, which can then be efficiently ligated under mild, aqueous conditions. The ability to start with cleaner inputs for the ligation step dramatically reduces the accumulation of byproducts throughout the overall process, simplifying subsequent purification demands and lowering solvent and energy consumption in downstream processing. Also, for highly modified constructs, ligation can simplify production by eliminating the need to repeatedly adjust protection strategies or work within the limitations of solid-phase chemistry, resulting in a more agile and resource-efficient process development.
Significant advancements in enzyme engineering underpin the success and versatility of these enzymatic approaches. Specialized platforms play a crucial role in developing tailored enzymes specifically optimized for RNA manufacturing. These highly engineered enzymes exhibit tailored substrate specificity, robust catalytic performance across a broad range of process conditions (e.g., temperature and pH) and remarkable tolerance for chemically modified bases and high-concentration substrates. This optimization minimizes unwanted side reactions and sustainably supports scalable, GMP-ready production. As an example, customized ligases can be developed to meet the specific demands of different RNA modalities, enabling reliable ligation even with challenging structural features or chemically modified junctions.
The broader sustainability and operational benefits of enzymatic synthesis are multifaceted. The most impactful is the drastic reduction in waste generation, primarily due to the elimination of large volumes of hazardous organic solvents. This directly translates to a lower PMI and a significantly smaller environmental footprint. Additionally, the aqueous nature of the process leads to an improved safety profile for manufacturing personnel and reduces the need for costly, specialized infrastructure. The cleaner synthesis also results in simplified downstream processing, requiring fewer and less intensive purification steps, which in turn reduces solvent and energy consumption. Lastly, the solution-phase format of enzymatic synthesis is inherently more amenable to continuous manufacturing processes, a future direction that promises even greater resource efficiency and minimal waste. By embracing these enzyme-enabled solutions, the biopharmaceutical industry can adopt a more sustainable and environmentally friendly approach to RNA manufacturing, ultimately contributing to a healthier planet and a more responsible future for medicine.
Ultimately, the enhanced efficiency and sustainability of enzymatic synthesis result in significant cost-effectiveness. By drastically reducing the reliance on expensive and hazardous organic solvents, minimizing chemical waste disposal and simplifying purification requirements, the operational costs associated with RNA manufacturing are substantially lowered (5, 9). Additionally, the higher yields and accelerated production timelines contribute to a more efficient use of resources and capital, ultimately making the production of RNA therapeutics more economically viable and accessible on a global scale. This holistic improvement across scalability, purity, timelines and cost positions enzymatic synthesis for the sustainable and widespread adoption of RNA-based medicines (5, 9).
Integration, innovation and regulatory alignment
The trajectory of RNA therapeutics manufacturing is undeniably shifting, with enzyme-enabled synthesis set to define its future. This evolving landscape will be characterized by a dynamic interplay of integration, continuous innovation and crucial regulatory alignment.
One prominent aspect of this future is the potential for hybrid approaches. While enzymatic synthesis offers compelling advantages, it is not necessarily an “either/or” proposition with SPOS. Strategic combinations, such as utilizing SPOS for the efficient synthesis of short, simple fragments followed by enzymatic ligation for assembly into complex, longer constructs, could leverage the strengths of both technologies. This flexibility allows manufacturers to tailor workflows to the specific requirements of each RNA molecule, optimizing for efficiency, cost and sustainability.
Continuous advancements in enzyme engineering and process development will continue to be a key driver. Specially created platforms will continue to refine enzymes for even greater catalytic efficiency, broader substrate specificity and enhanced tolerance to diverse RNA modifications. This ongoing innovation will expand the scope of what is feasible in RNA manufacturing, enabling the efficient production of increasingly complex RNA modalities, including site-specifically conjugated RNAs, highly modified duplexes and those requiring precise stereochemical control. The development of predictive tools and high-throughput screening for optimal enzyme-substrate pairings will further accelerate process development and reduce the risk associated with scale-up.
Crucially, the industry will see increasing emphasis on regulatory considerations and their adoption. As sustainability becomes a board-level priority and ESG metrics gain prominence, regulatory bodies are also adapting. The European Medicines Agency (EMA) has issued draft guidance on the development and manufacture of oligonucleotides, indicating a growing emphasis on process efficiency and impurity profiles. While specific sustainability regulations for oligonucleotide manufacturing are still evolving, the inherent green chemistry principles of enzymatic synthesis — reduced hazardous waste, lower energy consumption and safer reagents — align well with global trends toward greener pharmaceutical production. Collaborative partnerships between specialized technology providers and drug developers will be crucial to facilitating the adoption of these novel manufacturing platforms, ensuring a seamless transition and accelerating the path to market for next-generation RNA therapies. This collective effort will not only advance the science but also ensure that RNA therapeutics can be produced responsibly and be accessible for a global patient population.
References and notes
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- Agilent Technologies Inc. Agilent investing $725 million to expand state-of-the-art manufacturing capacity for production of nucleic acid-based therapeutics (Internet). Santa Clara (CA): Agilent Technologies Inc.; 2023 Jan 9 (cited 2025 Oct 3). Available from: https://www.investor.agilent.com/news-and-events/news/news-details/2023/Agilent-Investing-725-Million-to-Expand-State-of-the-Art-Manufacturing-Capacity-for-Production-of-Nucleic-Acid-Based-Therapeutics/default.aspx
- Wiegand DJ, Rittichier J, Meyer E, Lee H, Conway NJ, Ahlstedt D, et al. Template-independent enzymatic synthesis of RNA oligonucleotides. Nat Biotechnol. 2024 Jul 12;43(5):762–72. doi: 10.1038/s41587-024-02244-w. PMID: 38997579; PMCID: PMC12084152.
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- Verardo D, Adelizzi B, Horgan A, et al. Multiplex enzymatic synthesis of DNA with single-base resolution. Sci Adv. 2023 Jul 7;9(27):eadi0263. doi: 10.1126/sciadv.adi0263. PMCID: PMC10328407.
- Lund S, Potapov V, Johnson SR, Buss J, Tanner NA. Highly parallelized construction of DNA from low-cost oligonucleotide mixtures using Data-Optimized Assembly Design and Golden Gate. ACS Synth Biol. 2024 Mar 15;13(3):745–51. doi: 10.1021/acssynbio.3c00694. Epub 2024 Feb 20. PMID: 38377591; PMCID: PMC10949349.
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