The enzymatic advantage: Scaling RNA manufacturing for the next wave of therapeutics

RNA-based drugs, once confined to niche applications, are rapidly gaining mainstream adoption. Therapies targeting high-burden conditions, such as high cholesterol and cardiovascular disease, are creating a monumental shift, pushing the industry to scale production from small, specialized batches to metric tons of material annually. This massive increase in demand is straining traditional manufacturing methods and driving the urgent need for more efficient, sustainable, and cost-effective solutions.

At the forefront of this manufacturing evolution is enzyme-enabled synthesis. Leveraging the precision of biological catalysts will be the key to unlocking the full potential of therapeutic oligonucleotides, including small interfering RNA (siRNA), including guide RNA (gRNA), and antisense oligonucleotides (ASOs).

Why traditional RNA manufacturing is falling short

The current gold standard, solid-phase oligonucleotide synthesis (SPOS), was initially developed for small-scale synthesis (<10 kg). While adequate for early-stage development and orphan indications, it’s proving difficult to adapt for the metric-ton demands of therapies targeting millions of patients. The limitations of SPOS are rooted in its chemistry:

• Poor scalability
  The stepwise, surface-bound nature of the synthesis process limits the amount of material that can be produced in a single batch.
• Challenges in achieving quality standards

Achieving the required high quality for RNA therapies with SPOS workflows involves multiple, resource-intensive purification steps, which add costs, generate hazardous waste and reduce speed to market.

• High cost and low yield
  The harsh chemical conditions cause cumulative yield loss as the RNA sequence lengthens, significantly increasing both production costs and complexity.
• Environmental strain
  SPOS workflows heavily rely on large volumes of hazardous organic solvents, such as acetonitrile, toluene and dichloromethane. This presents significant safety and environmental challenges, including complex waste management and recovery, and necessitates extensive, specialized infrastructure.

The approval of high-volume therapies, such as the cholesterol-lowering drug inclisiran, has made it clear that a manufacturing pivot is essential for the future of RNA therapeutics.

The efficiency and sustainability of enzyme-enabled processes

Enzyme-enabled approaches offer a fundamentally superior path for manufacturing therapeutic RNA. They leverage the precision and efficiency of biological catalysts, transforming a complex chemical process into a streamlined, biology-inspired one.

1. Water-based, greener chemistry

Unlike traditional methods, enzymatic processes occur in water-based solutions. This eliminates the need for toxic organic solvents, significantly reducing the overall environmental footprint and simplifying the production workflow. This alignment with greener, more sustainable manufacturing practices is crucial for the pharmaceutical industry’s future.

2. Streamlined production and lower costs

Enzyme-based synthesis simplifies several complex, costly steps associated with SPOS, such as:

• Eliminating protecting groups: Biological systems are inherently selective, reducing the need for extensive chemical protecting groups.
• Simplified purifications: Reduced impurities simplify downstream purification, a major cost driver in traditional synthesis.

3. Modular assembly for complex constructs

Enzymatic ligation (the joining of shorter RNA fragments into full-length sequences) offers a decisive advantage, particularly for the complex, highly modified siRNA, gRNA and ASO constructs being developed today, including:

• Reduced impurities: Ligation minimizes the exposure of the full-length product to the harsh, repeated conditions of linear synthesis.
• Modular scalability: Different RNA fragments can be synthesized in parallel and then efficiently joined, supporting rapid, modular, and high-volume assembly.

Enzyme engineering: The engine of innovation

The true game-changer has been the advancements in enzyme engineering. By optimizing enzymes for stability, sequence promiscuity, and high-conversion efficiency, manufacturers gain unprecedented control and flexibility. Engineered enzymes offer many key benefits for drug developers:

• Versatility
  Optimized enzymes can work on a broad spectrum of RNA sequences without extensive process re-engineering.
• Precision
  Enzymes naturally exhibit exquisite stereochemical control, opening opportunities to define structural features that can enhance drug efficacy [1].
• Expanded scope
  Milder water-based reaction conditions enable the successful synthesis of oligo modifications that are challenging or incompatible with harsh SPOS chemistry.

This increased flexibility and the ability to manufacture highly modified, high-quality oligonucleotides at scale translate into reduced costs and faster development timelines.

The future of flexibility, scalability and sustainability

As RNAi therapeutics expand into chronic, high-dose disease indications that require metric tons of material annually, the industry’s ability to produce high-quality RNA oligonucleotides will be the ultimate differentiator.

The most significant opportunity for innovation lies in flexible, scalable, and sustainable manufacturing. Enzyme-based approaches are uniquely positioned to meet this demand, offering a path to cost reduction, streamlined processes and higher yields, faster scale-up and reduced environmental impact. By embracing these enzymatic technologies, the industry can confidently move beyond the limitations of legacy chemistry and unlock the full therapeutic potential of RNA medicine.

Reference

1. https://www.nature.com/articles/s41419-022-05075-2