Accelerating Scalable and Sustainable siRNA Manufacturing with Ligation-based Synthesis

The phenomenal growth of RNA interference (RNAi) therapeutics, with hundreds of oligonucleotide assets in the pipeline and a projected market value increase from $2.6 billion to $6.6 billion by 2030, is creating unprecedented pressure on manufacturing capacity [1]. Meeting this need requires innovative solutions that move beyond the scale and capacity limitations of traditional solid-phase oligonucleotide synthesis (SPOS) methods. Enzymatic ligation of oligonucleotide fragments has emerged as a crucial technology to achieve scalable, cost-efficient and sustainable RNA production.

Why is Enzymatic Ligation Efficient?

The core principle of ligation is simple but powerful. Instead of synthesizing a long siRNA molecule in a single lengthy process, the sequence is broken into shorter fragments, which are then enzymatically joined. This approach fundamentally improves manufacturing economics and performance:

• Improved yield and purity
  Synthesizing shorter fragments inherently leads to better purity and yield than attempting a full-length synthesis, where the cumulative loss from coupling inefficiencies compounds across the entire strand. Ligation leverages this high-quality input to achieve a higher overall product yield.
• Cost reduction and scalability
  Ligation reduces manufacturing complexity and eliminates or streamlines purification steps typically associated with full-length synthesis, reducing the cost of goods sold (COGS). When the full sequence is split into multiple short fragment syntheses (sequential SPOS), the shorter length of each fragment drastically reduces the cumulative failure rate and the accumulation of truncated impurities that plague long SPOS runs. This reduction in risk means the process is more reliable, leading to fewer failed batches and a higher-quality intermediate, which offers improved scalability and simplifies the subsequent purification of the final, full-length product.
• Enhanced sustainability
  Ligation helps shift the manufacturing process away from the extensive use of harmful organic solvents characteristic of SPOS (which can require over 3,000 kg of acetonitrile per kilogram of the active pharmaceutical ingredient) toward partially or fully water-based systems, significantly improving the environmental footprint.

Driving Performance with Engineered Enzymes and Fragment Design

The success of the ligation workflow can rely heavily on the catalyst: the ligase enzyme. Wild-type (WT) ligases often perform poorly when faced with the complex chemical modifications, such as 2′-O-methyl and 2′-fluoro groups, commonly used to enhance the stability and efficacy of siRNA therapeutics. Although a WT enzyme might eventually achieve a similar final conversion to an engineered counterpart, it will typically be less time-efficient and require greater resources and intensive downstream purification to compensate for lower efficiency and fidelity.

Highly engineered double-stranded RNA (dsRNA) ligases are explicitly designed to overcome these limitations, delivering performance metrics essential for commercial manufacturing:

• Speed and volumetric productivity
  Optimized enzymes significantly accelerate the process, achieving high conversion rates (e.g., >95%) much faster than WT counterparts. Crucially, they tolerate high substrate loads (up to 100 g/L), allowing for larger batch sizes and increased volumetric productivity.
• Process robustness
  Engineered ligases are highly robust, maintaining activity across broader temperature and pH ranges, which provides greater flexibility in process design and ease of transfer between manufacturing facilities.
• Impurity tolerance
  Some engineered enzymes exhibit tolerance to impurities in crude fragment pools, often enabling the ligation reaction to occur with minimal intermediate purifications.

Validating the Ligation Advantage: Clinical Case Studies

Moving from theory to practice, data from therapeutic compounds confirms that enzymatic ligation provides a reliable, high-fidelity path to complex RNA therapeutics. The real-world application of this technique highlights its dual benefit of process simplification and superior product quality. For specific sequences, we employ an immobilization technique that, when successfully implemented, meets the final drug substance’s rigorous standards for enzyme clearance.

“Purification by Ligation” Streamlines the Workflow

Superior product quality is achieved because the ligation step itself serves as an inherent purification mechanism. The enzyme-driven process selectively joins only high-quality, fully elongated fragments, effectively suppressing the incorporation of shorter, undesired impurities. This phenomenon directly contributes to the process simplification benefit by dramatically reducing the need for intensive pre-ligation purification steps.

Inclisiran Synthesis

The application of enzymatic ligation to the siRNA therapeutic, inclisiran, provides a clear example of its benefits. Data comparing short RNA fragments demonstrated that fragment quality is a critical quality attribute for maximizing ligation success [2]. For example, fragments with higher initial purity can result in a high-quality siRNA duplex without requiring post-ligation purifications [3].

Vutrisiran Scale-up and Rational Design

For another siRNA drug, vutrisiran, an advanced machine learning (ML) tool was used to rationally design and predict the optimal fragment designs (disconnection schemes) for maximal ligation efficiency. This data-driven approach achieved high ligation success (over 95%) and yielded material with excellent post-purification purity (over 98%). The technical feasibility of this route has been demonstrated by successfully scaling up the process to hundreds of micromoles, proving its applicability for large-scale manufacturing.

To ensure a safe and effective drug substance, a robust control strategy for residual enzyme is vital. Various techniques are utilized to facilitate the consistent removal of the ligase, including ligase immobilization and downstream purification methods (such as affinity and ion-exchange chromatography). Evaluating and demonstrating the efficient removal of the ligase is a key step in process development, which simplifies downstream processing and ensures that the final drug substance meets the required clearance targets.

The Future of High-purity RNA Manufacturing

The evidence is clear: enzymatic ligation of oligonucleotide fragments is a validated, commercially viable platform that fundamentally changes how complex RNA therapeutics are made. By dividing the synthesis challenge into manageable, high-purity fragment steps, manufacturers can maximize yield, minimize costs and future-proof their supply chains against the growing demand for scaling RNA therapeutics. This approach delivers the high yield, exceptional purity and manufacturability required to propel the next generation of RNA therapeutics, from siRNAs to more complex structures, into the clinic and onto the market.

To Learn More About the Future of RNA Manufacturing, Access the Full TIDES USA 2025 presentation today.

References

1. https://www.grandviewresearch.com/industry-analysis/rnai-technology-market-report
2. https://www.biospace.com/press-releases/codexis-unveils-pioneering-enzymatic-synthesis-data-to-enable-the-future-manufacturing-of-rnai-therapeutics
3. https://d1io3yog0oux5.cloudfront.net/_cb84afbad4863e8a8ee87f45d2aba1cf/codexis/db/1454/16519/file/TIDES_US_2025_052025_Final.pdf