By: David Butler, PhD

INTRODUCTION

Nucleic acid technologies (NAT) are now a central focus of modern drug development. The success of mRNA vaccines and the growing number of small interfering RNA (siRNA) and single guide RNA (sgRNA) drugs in clinical pipelines are reshaping how the industry targets disease. siRNA and sgRNA are oligonucleotide modalities that allow the selective silencing or editing of disease-associated genes, opening new therapeutic avenues across cardiovascular, metabolic and genetic disorders.

This momentum, however, brings new manufacturing challenges. Achieving the required levels of purity, consistency and cost-efficiency at a commercial scale is increasingly difficult using conventional methods. Solid-phase oligonucleotide synthesis (SPOS), long the industry standard, now faces limits in scalability, sustainability and yield.

To meet future demand, developers are turning to next-generation approaches. Among these, chemoenzymatic ligation offers a practical and scalable bridge between traditional chemical synthesis and fully enzymatic methods. By combining the precision of enzymatic assembly with the flexibility of SPOS fragment production, this hybrid process enables high-purity oligonucleotide manufacturing at scale, while eliminating many of the inefficiencies of full-length chemical synthesis. In this article, David Butler, Chief Technical Officer at Hongene Biotech, explains how chemoenzymatic ligation can be implemented at an industrial scale and what it means for the future of oligonucleotide manufacturing.

WHY TRADITIONAL SPOS STRUGGLES TO MEET FUTURE DEMAND

The phosphoramidite-based SPOS method has been the foundation of oligonucleotide synthesis for nearly four decades. It remains highly effective for short, chemically modified sequences, but as the length and volume of oligonucleotides increase, its limitations become evident. ¹

SPOS is a batch-based process, typically yielding up to 10 kg per run and consuming large volumes of organic solvents and reagents. Scaling up requires multiple synthesis campaigns and batch pooling, driving up cost, complexity and environmental burden. As production scales, so too do sustainability and regulatory concerns.

Issues with SPOS are often most keenly felt when synthesizing long single-stranded oligonucleotides such as sgRNA and pegRNA. Each nucleotide addition during chain elongation introduces the risk of incomplete coupling and side reactions, leading to the formation of truncated sequences (shortmers) and other impurities that can accumulate through the synthesis cycle. These byproducts are difficult to remove during chromatographic purification, resulting in poor yield and issues with batch-to-batch consistency.

Market demand adds urgency. siRNA drugs targeting genes associated with cardiometabolic disease, such as PCSK9, AGT, LPA, HSD, APOC3 and INHBE, are advancing toward large patient populations, with projected global needs in the multi-ton range. SPOS, in its current form, is unlikely to support this level of commercial-scale output efficiently.

To meet this challenge, the industry needs platforms that preserve the flexibility of chemical synthesis while enabling scalable, cost-effective and environmentally sustainable production.

THE EVOLUTION OF OLIGONUCLEOTIDE MANUFACTURING

Oligonucleotide manufacturing can be described in terms of three generations of technology.

• Generation 1: Chemical synthesis. Traditional SPOS remains the industry standard for short chemically modified sequences. However, its limitations in scalability, yield and environmental impact become increasingly apparent as demand grows.
• Generation 2: Chemoenzymatic ligation. A hybrid approach that begins with the synthesis of short oligonucleotide fragments using SPOS. These fragments are then enzymatically ligated to produce full-length sequences, delivering improved yield, reduced impurities and better process efficiency than full-length SPOS.
• Generation 3: Enzymatic synthesis. A next-generation strategy that uses modified nucleoside triphosphates (NTPs) and engineered enzymes to build oligonucleotides, emulating cellular processes of DNA and RNA polymerization. Whilst not ready for industrial use, recent reports have demonstrated the potential of the technology to synthesize DNA2 and chemically modified RNA.^3,4

Chemoenzymatic ligation stands out because it can be implemented today using established SPOS infrastructure to synthesize high-purity fragments with fewer chances for impurity formation. These fragments are then joined enzymatically, creating complete oligonucleotide products without the inefficiencies of full-length SPOS. The result is a scalable, modular and environmentally responsible process that fits seamlessly into current manufacturing environments.

HOW CHEMOENZYMATIC LIGATION WORKS

Chemoenzymatic ligation combines the flexibility of chemical synthesis with the precision of enzymatic assembly. Short oligonucleotide fragments, also called blockmers, are synthesized by SPOS, purified, then joined in solution by ligase enzyme,⁵ which catalyzes the formation of phosphodiester bonds between the terminal 3′-hydroxyl and 5′-phosphate groups of adjacent fragments.

A key strength of this approach lies in its selectivity. Only fragments with correctly positioned terminal groups serve as substrates, meaning that many of the truncated impurities that are common in SPOS are excluded from the final product, reducing the purification burden and improving final purity.

The ligation reactions are carried out in aqueous solution at concentrations up to 120 g/L, with parameters such as pH and temperature finely tuned to optimize efficiency and fidelity. Single-use bioreactors and batch reactors enable scalability within standard GMP manufacturing suites.

Two chemoenzymatic ligation strategies have been the focus of development at Hongene (figure 1):

Sticky end ligation – Generally used for double-stranded oligonucleotides such as siRNA, this method uses complementary overhangs to align fragments before ligation.^6-8

Splinted ligation – Applied to single-stranded molecules such as sgRNA and pegRNA, this approach uses short complementary DNA “splints” to align fragments and guide enzymatic assembly.^9,10

Both processes are compatible with the chemical modifications widely used in therapeutic oligonucleotide production, including backbone modifications (phosphodiester, phosphorothioate, mesylphosphoramidate), ribose modifications (2′-O-methyl, 2′-fluoro) and conjugates such as GalNAc. As a result, chemoenzymatic ligation maintains compatibility with current drug designs while enabling the efficient manufacture of longer, more complex oligonucleotide molecules.

Both sticky-end and splinted ligation processes are also highly scalable. Because ligation occurs in solution, the process can be run in stainless steel or single-use bioreactors, enabling straightforward scale-up using equipment common to manufacturers. Yields are typically higher than full-length SPOS, as shorter fragments are easier to synthesize and purify and the enzymatic ligation step proceeds with near-quantitative conversion under mild, aqueous conditions. This results in higher purity, reduced solvent usage and a smaller environmental footprint, supporting more sustainable and cost-effective oligonucleotide manufacturing.

INNOVATION DRIVING THE NEXT PHASE OF MANUFACTURING

Chemoenzymatic ligation is a continuously evolving platform. Ongoing research is focused on enhancing scalability, reducing environmental impact and driving down cost. Current efforts target three core innovation levers:

Scalable fragment synthesis: Transitioning from flow-through synthesizers to batch or liquid-phase synthesis methods will increase capacity and reduce solvent use, making the system more compatible with large-scale manufacturing.

Elimination of chromatography: New workflows are being developed to bypass purification column chromatography. Early results suggest that “crude-to-purified” and even “crude-to-crude” ligation strategies can maintain product quality while significantly reducing material use and cost.

Engineered ligases: Thermostable ligases are being developed to operate efficiently at elevated temperatures. Conducting the reaction at higher temperatures reduces RNA secondary structure formation, which is hypothesized to improve ligation efficiency by enhancing the enzyme’s ability to recognize and join fragments that are correctly aligned.

These innovations are not only advancing chemoenzymatic ligation but also laying the foundation for next-generation fully enzymatic RNA synthesis. As the field progresses, hybrid platforms will serve as a critical bridge between today’s manufacturing needs and tomorrow’s biologically inspired production systems.

REGULATORY READINESS & CMC ALIGNMENT

As manufacturing technologies for oligonucleotides change, regulatory expectations are also advancing in parallel. With chemoenzymatic ligation approaching wider clinical and eventually commercial application, clear global guidance is needed to define acceptable CMC practices.

One important regulatory consideration is how oligonucleotide fragments are classified within the manufacturing process, either as starting materials or GMP intermediates. This designation defines the level of GMP oversight and documentation required and is made in accordance with the principles outlined in ICH Q11.

Analytical comparability is another priority. Developers must demonstrate that the quality of ligation-derived oligonucleotides is well-controlled and comparable to those produced via traditional SPOS. Comprehensive analytical characterization, including impurity profiling and stereochemical analysis, supports this and helps de-risk regulatory submissions.

Analytical testing strategies must evolve to accommodate the chemoenzymatic manufacturing approach. For siRNA products, the sense and antisense strands are no longer synthesized separately, making individual-strand testing unfeasible. Instead, quality is assessed at the fragment level, with denaturing HPLC/MS methods used to identify and quantify single-strand components and their associated impurities within the duplex context.

Phosphorothioate stereochemistry is an increasingly scrutinized quality attribute in therapeutic oligonucleotide manufacturing. Differences in reaction conditions between SPOS and ligation-based processes can influence the stereochemical distribution of the final product. HPLC and nuclear magnetic resonance (NMR) methods are used for demonstrating batch-to-batch consistency and establishing comparability between synthetic approaches.

The enzyme-based nature of chemoenzymatic ligation introduces a new critical quality attribute: residual enzyme content. Ensuring that enzymes used during ligation are of acceptable quality and effectively removed is essential. Trace enzyme levels can be detected and quantified using methods such as ELISA and MS to confirm compliance with established specifications.

TOWARD A SCALABLE & SUSTAINABLE OLIGONUCLEOTIDE MANUFACTURING ECOSYSTEM

Chemoenzymatic ligation represents a shift toward sustainable and scalable oligonucleotide manufacturing. By combining the precision of enzymatic ligation with the versatility of SPOS, this hybrid approach offers a practical path to consistent, cost-effective and environmentally responsible manufacturing. It enables developers to meet rising global demand without compromising on quality, control or speed.

A vertically integrated model, like that at Hongene, linking raw material production, ligation platform technology and CDMO services for both drug substance and drug product, addresses the growing need for supply chain cohesion and resilience. This approach can be a key competitive advantage.

Looking ahead, continued innovation in chemoenzymatic manufacturing is expected to strengthen process robustness and scalability, shaping how broadly chemoenzymatic assembly can be applied across different NAT applications. The focus now is on translating this manufacturing technology to support global therapeutic development and ensure patients have access to the life-changing medicines they need.

REFERENCES

1. Sanghvi, Y. S.; Ferrazzano, L.; Cabri, W.; Tolomelli, A. Sustainable Approaches in Solid-Phase Oligonucleotide Synthesis: Current Status and Future Directions. In Sustainability in Tides Chemistry; Tolomelli, A., Ferrazzano, L., Cabri, W., Eds.; Royal Society of Chemistry, 2024; pp 228-247. https://doi.org/10.1039/9781837674541-00228.
2. Forget, S. M.; Krawczyk, M. J.; Knight, A. M.; Ching, C.; Copeland, R. A.; Mahmoodi, N.; Mayo, M. A.; Nguyen, J.; Tan, A.; Miller, M.; Vroom, J.; Lutz, S. Evolving a Terminal Deoxynucleotidyl Transferase for Commercial Enzymatic DNA Synthesis. Nucleic Acids Res. 2025, 53 (4), gkaf115. https://doi.org/10.1093/nar/gkaf115.
3. Wiegand, D. J.; Rittichier, J.; Meyer, E.; Lee, H.; Conway, N. J.; Ahlstedt, D.; Yurtsever, Z.; Rainone, D.; Kuru, E.; Church, G. M. Template-Independent Enzymatic Synthesis of RNA Oligonucleotides. Nat. Biotechnol. 2025, 43 (5), 762-772. https://doi.org/10.1038/s41587-024-02244w.
4. Moody, E. R.; Obexer, R.; Nickl, F.; Spiess, R.; Lovelock, S. L. An Enzyme Cascade Enables Production of Therapeutic Oligonucleotides in a Single Operation. Science 2023, 380 (6650), 1150-1154. https://doi.org/10.1126/science.add5892.
5. Nandakumar, J.; Shuman, S.; Lima, C. D. RNA Ligase Structures Reveal the Basis for RNA Specificity and Conformational Changes That Drive Ligation Forward. Cell 2006, 127 (1), 71-84. https://doi.org/10.1016/j.cell.2006.08.038.
6. Sosic, A.; Pasqualin, M.; Pasut, G.; Gatto, B. Enzymatic Formation of PEGylated Oligonucleotides. Bioconjug. Chem. 2014, 25 (2), 433-441. https://doi.org/10.1021/bc400569z.
7. Khorana, H. G. Nucleic Acid Synthesis. Pure Appl. Chem. 1968, 17 (3), 349-382. https://doi.org/10.1351/pac196817030349.
8. Paul, S.; Gray, D.; Caswell, J.; Brooks, J.; Ye, W.; Moody, T. S.; Radinov, R.; Nechev, L. Convergent Biocatalytic Mediated Synthesis of siRNA. ACS Chem. Biol. 2023. https://doi.org/10.1021/acschembio.3c00071.
9. Moore, M. J.; Query, C. C. [7] Joining of RNAs by Splinted Ligation. In Methods in Enzymology; RNA – Ligand Interactions, Part A; Academic Press, 2000; Vol. 317, pp 109-123. https://doi.org/10.1016/S00766879(00)17009-0.
10. Bigatti, M.; Moser, A.; Dierssen, B.; Frrokaj, S.; Covato, E.; Pfleger, C.; Lill, J.; Leiser, Y.; Zuber, J.; Staempfli, A.; Sladojevich, F.; Koenig, S. G. Development of a Broadly Applicable Enzymatic Ligation Process for the Production of Single Guide RNAs. Org. Process Res. Dev. 2025, 29 (5), 1228-1236. https://doi.org/10.1021/acs.oprd.4c00502.