Developing RNA therapeutics requires years of scientific innovation and dedicated investment. However, advancing a promising preclinical candidate into clinical development—and ultimately commercial production—presents a major manufacturing challenge: scalability. Manufacturers must be able to increase production from grams to kilograms or even metric tons while maintaining product quality, batch consistency, and cost-effectiveness.
At this critical stage, conventional manufacturing approaches may become a bottleneck. Solid-phase oligonucleotide synthesis (SPOS), the established method for oligonucleotide production, is widely used in research and clinical manufacturing. However, its stepwise synthesis process presents increasing challenges in yield, impurity control, resource consumption, and manufacturing efficiency as oligonucleotide length and production volumes increase.
These challenges have driven growing interest in alternative manufacturing strategies, including chemoenzymatic ligation, which combines chemical synthesis with enzymatic reactions to support the production of increasingly complex RNA therapeutics at scale.
1. Limitations of Conventional Oligonucleotide Synthesis
SPOS involves sequentially adding nucleotide building blocks to a growing chain through repeated chemical coupling cycles. Although this approach is well established and highly effective for many oligonucleotide applications, several limitations become more pronounced when synthesizing longer sequences or increasing production volumes. The limitations include:
(1) Cumulative synthesis losses and impurity formation: Each coupling cycle carries a small risk of incomplete reactions and unwanted side reactions. As the number of synthesis steps increases, these losses accumulate, reducing the proportion of full-length product (FLP) and increasing the complexity of impurity profiles. For longer molecules, such as single-guide RNA (sgRNA), controlling and removing truncated sequences and other process-related impurities can become particularly challenging.
(2) Reduced overall yield and increased manufacturing costs: As mentioned previously, incomplete coupling can generate truncated products that must be separated from the desired full-length sequence. If oligonucleotide length increases, the cumulative impact of incomplete reactions can reduce overall FLP yield, increasing purification requirements, raw material consumption, waste generation, and manufacturing costs.
(3) Challenges in scaling production capacity: Conventional flow-through oligonucleotide synthesizers operate within defined reactor and equipment capacities. Increasing output therefore often requires additional synthesis equipment and production campaigns rather than simply expanding the capacity of an individual process. This can increase facility requirements, operational complexity, and manufacturing costs when meeting projected large-scale demand.
2. Chemoenzymatic Ligation: Combining Chemical and Enzymatic Synthesis
Chemoenzymatic ligation is a hybrid manufacturing approach. Rather than synthesizing an entire long sequence, the target oligonucleotide is divided into shorter fragments. These fragments are independently manufactured using SPOS and subsequently assembled through an enzyme-catalyzed ligation reaction.
This modular strategy offers several potential advantages for the manufacturing of complex RNA therapeutics:
(1) Improved product quality and impurity control: Shorter oligonucleotide fragments have higher yields and simpler impurity profiles than their corresponding full-length sequences synthesized directly by SPOS. During ligation, the enzyme recognizes specific terminal structures, typically requiring a 5’-phosphate group and a 3’-hydroxyl group on the appropriate substrates. This substrate selectivity can help limit the incorporation of improperly configured fragments and improve the overall quality of the assembled product.
(2) Higher overall manufacturing efficiency: By reducing the need for extensive stepwise chemical synthesis of long sequences, chemoenzymatic ligation has the potential to lower reagent and solvent consumption. Combined with improved yields and optimized purification processes, this may reduce chemical waste and the environmental footprint of oligonucleotide manufacturing.
(3) Scalability for large-volume production: Enzymatic ligation is performed in solution and can be adapted to larger-scale batch reactors. This provides greater flexibility in manufacturing infrastructure and offers a potential pathway toward higher production volumes within established GMP facilities.
3. Tailored Ligation Strategies for Different RNA Modalities
Hongene has developed distinct ligation strategies to accommodate different oligonucleotide formats and structural requirements:
(1) double-stranded RNA: For double-stranded small interfering RNA (siRNA), Hongene has advanced sticky-end ligation into GMP manufacturing facilities to support clinical-stage programs. This approach uses complementary overhanging sequences to facilitate the alignment and joining of oligonucleotide fragments.
(2) single-stranded RNA: For longer single-stranded molecules, such as sgRNA used in gene-editing applications, Hongene employs splinted ligation. In this approach, a complementary splint strand aligns adjacent oligonucleotide fragments, positioning their termini for enzymatic joining.
These complementary strategies demonstrate the adaptability of chemoenzymatic ligation across different RNA formats and therapeutic applications.
For a more detailed explanation of sticky-end ligation and splinted ligation, download Hongene’s Chemoenzymatic Ligation White Paper.
4. Addressing CMC and Regulatory Considerations
As chemoenzymatic ligation advances into clinical and commercial manufacturing, establishing robust Chemistry, Manufacturing, and Controls (CMC) strategies is essential. Hongene has developed control strategies to address both established oligonucleotide Critical Quality Attributes (CQAs) and process-specific considerations, including:
(1) Starting material designation: Establishing the regulatory classification and appropriate control strategy for chemically synthesized oligonucleotide fragments used as starting materials, in alignment with relevant regulatory expectations.
(2) Phosphorothioate stereochemistry: Developing analytical methods to characterize and control phosphorothioate (PS) stereochemistry, supporting consistent product quality and comparability with conventionally synthesized materials where applicable.
(3) Residual enzyme control: Establishing analytical methods to detect and quantify residual ligase enzymes, alongside appropriate safety assessments to evaluate potential risks associated with residual process-related proteins.
Through these CMC development efforts, Hongene is working to establish chemoenzymatic ligation as a robust manufacturing platform suitable for clinical development and future commercial applications.
Advancing Scalable and Sustainable RNA Manufacturing
As the pipeline of RNA therapeutics expands, manufacturing technologies must evolve to support increasingly complex molecules and growing commercial demand.
Looking ahead, fully enzymatic synthesis (3rd Generation) may offer further opportunities to transform oligonucleotide manufacturing. In the meantime, chemoenzymatic ligation (2nd Generation) continues to advance through process optimization and technology development.
Hongene is exploring several approaches to further improve manufacturing efficiency, such as using crude fragments for ligation to further increase the yield, as well as enzyme engineering to expand substrate compatibility and improve ligation performance under different reaction conditions, e.g., elevated temperatures.
To learn more about how Hongene can support your RNA therapeutic manufacturing program, please contact our team.