Chemoenzymatic Ligation Emerges as Scalable Solution to Nucleic Acid Therapeutics Manufacturing Bottlenecks

The success of mRNA vaccines during the COVID-19 pandemic has highlighted the broader potential of nucleic acid therapeutics (NATs), a class that includes siRNA, antisense oligonucleotides, guide RNAs for gene editing, and increasingly sophisticated mRNA constructs for oncology, rare disease, and infectious disease. Yet this momentum has exposed a fundamental tension in the sector: discovery capabilities are outpacing the industry's ability to manufacture these modalities at scale. As Dr. Lena Preuß, Technical Sales Specialist at Hongene Biotech (搜索)搜索该词条, notes, the focus is increasingly shifting toward overcoming large-scale manufacturing bottlenecks, particularly for the single-guide RNA (sgRNA) components that direct the Cas9 (搜索)搜索该靶点 nuclease to its genomic target.

The Limits of Solid-Phase Oligonucleotide Synthesis

Solid-phase oligonucleotide synthesis (SPOS) has been the standard manufacturing method for oligonucleotides for four decades and works effectively for short, chemically modified sequences. However, as the field moves toward large-patient indications with more complex molecules, its operational limitations become apparent. Flow-through synthesizers employed in SPOS are typically limited to batches of around 10 kg, meaning larger quantities require running multiple campaigns and pooling batches in a "scale-out" approach that drives up cost, complexity, and waste.

For siRNA drugs targeting cardiometabolic diseases, where global patient populations can number in the tens of millions, demand for a single successful product could eventually reach ton-scale API requirements annually — a scale SPOS was never designed to meet. Long oligonucleotides, such as guide RNA molecules exceeding 100 nucleotides in length, present additional problems. Sequential phosphoramidite coupling means impurities accumulate with every step, resulting in truncated sequences and other byproducts that are difficult to remove during purification and challenging batch-to-batch consistency.

The primary challenge with SPOS is the cumulative risk associated with stepwise addition of nucleotides. Each addition carries a small probability of an error or side reaction, and for longer molecules like sgRNA, these errors accumulate significantly, forming (n-1) deletion sequences and other impurities that are chemically similar to the target drug substance and exceptionally difficult to remove. The high solvent use and reagent consumption of traditional SPOS also present growing environmental, social, and governance (ESG) concerns.

Chemoenzymatic Ligation as a Second-Generation Technology

Chemoenzymatic ligation addresses these constraints by combining chemical and enzymatic synthesis. Rather than building the full-length oligonucleotide in one synthesis campaign, the molecule is assembled from shorter SPOS-derived fragments — known as blockmers — that are joined using T4 RNA ligase (搜索)搜索该靶点. The shorter fragments are synthesized more cleanly, and the ligation reaction is highly selective, proceeding preferentially with correctly positioned, viable substrates. Many fragment impurities are therefore excluded from the final product, reducing truncation sequences, simplifying downstream processing, and enhancing purity.

The ligation step is performed in aqueous buffer, making it compatible with batch reactors and single-use bioreactors, which are inherently more scalable than flow-through synthesizers. The industry is currently exploring two distinct workflows: sticky end ligation for double-stranded molecules such as siRNA, and splinted ligation for single-stranded oligonucleotides such as guide RNAs. Both approaches support the full range of chemical modifications found in NAT development candidates, including phosphorothioate, phosphodiester, and phosphoramidate backbones, 2′-O-methyl and 2′-fluoro ribose modifications, and GalNAc conjugates.

The ligase enzymes used in enzymatic ligation are highly substrate-specific, typically accepting only fragments with correctly positioned 5'-phosphate and 3'-hydroxyl groups, which helps ensure that common deletion impurities are rejected and purged during downstream processing. The solution-based ligation reaction is ideally suited for large-scale stainless steel batch reactors and can be readily integrated into existing good manufacturing practice (GMP) infrastructure.

Flexibility in Purity, Yield, and Cost

The chemoenzymatic ligation process offers flexibility in how manufacturers balance purity, yield, and cost. Fragments sufficiently pure in crude form can be processed by ultrafiltration and diafiltration without chromatography, increasing yield without sacrificing purity — a workflow called C-to-P (crude-to-purified). This lays the foundation for a fully chromatography-free C-to-C (crude-to-crude) process, described as a potentially game-changing advance for high-volume cardiometabolic programs where cost of goods is a key commercial consideration.

Realizing the full potential of this approach requires a more robust enzymatic toolkit. Wild-type T4 RNA ligase (搜索)搜索该靶点 has limitations around operating temperature, and RNA substrates can form secondary structures that reduce ligation efficiency. The development of thermostable T4 RNA ligase variants that function at temperatures up to 57°C can help reduce secondary structure formation, improving substrate alignment and ligation efficiency. This advance is expected to be particularly beneficial for enabling C-to-C processes and for synthesizing very long constructs such as prime editing guide RNA (pegRNA).

Optimizing mRNA Performance Through Cap Analog Chemistry

Innovation in NAT manufacturing extends beyond oligonucleotides. In the mRNA field, considerable effort is focused on optimizing the 5′ cap structure, which directly influences in vitro transcription (IVT) performance, translation efficiency, transcript stability, and innate immune recognition. The conventional Cap1 structure, which includes a 2′-O-methylation at the first nucleotide (N1), has known limitations, including the ability of the innate immune protein IFIT1 (搜索)搜索该靶点 to bind Cap1 RNA under inflammatory conditions, sterically blocking translation initiation.

Novel cap analogs address this through rational structure-based design. By substituting a bulkier 2′-O-ethyl group at the N1 ribose position, the cap introduces a steric clash with key IFIT1 (搜索)搜索该靶点 residues, destabilizing the IFIT1-RNA interaction while maintaining compatibility with standard IVT workflows. Both trinucleotide Cap1 analogs and tetranucleotide variants have been developed, improving capping efficiency and broadening applicability across mRNA chemistries.

In IFNα-stimulated cell models and LPS-challenged mice, these novel analogs maintain protein expression at levels that conventional cap structures cannot match under the same conditions. In a melanoma (搜索)搜索该疾病 tumor inhibition model, mRNA using this modified cap structure achieved approximately 60 percent tumor growth inhibition versus around 30 percent for a conventional Cap1 comparator. The recent advance of mRNA compositions containing one of these novel cap analogs into investigator-initiated clinical trials for solid tumors, acute myeloid leukemia (搜索)搜索该疾病, and post-surgical pancreatic cancer (搜索)搜索该疾病 represents an important translational milestone.

The Vulnerability of Fragmented Supply Chains

Phosphoramidites, loaded solid supports, GalNAc, enzymes, nucleotide triphosphates, and cap analogs are complex inputs whose quality directly impacts NAT drug substance quality and therapeutic success. Even trace impurities in phosphoramidites can react, propagate, and accumulate during SPOS, producing a distribution of related impurities in the oligonucleotide product. Controlling these impurities to ensure batch-to-batch consistency requires institutional knowledge, combined with robust processes and dedicated infrastructure.

Despite this, it is typical for NAT developers to source individual raw materials from multiple vendors, distributing quality risk across organizations and requiring time-consuming material handoffs. A vertically integrated supply model consolidates the supply chain from nucleoside building blocks through to cGMP drug substance and drug product, reducing handoffs and concentrating accountability. When a raw material or process changes, the investigation, corrective action, and revalidation all happen within one organization, removing the delays and misalignment that characterize fragmented supply chains.

Advancing Chemistry and Infrastructure in Tandem

The NAT field is not short of promise: siRNA programs are advancing toward commercial volumes that will test existing infrastructure; gene editing medicines are moving into the clinic with increasingly complex guide RNA constructs; and mRNA therapeutics are demanding improved performance, including sustained protein expression in immunologically challenging environments. However, developers are still largely using a manufacturing toolkit designed for a previous era.

Chemistry innovation and manufacturing capability must develop together. Innovation without infrastructure stays in the lab, and infrastructure without innovation delivers the same products without improvements in quality or efficiency. The next generation of NATs will be defined by organizations that invest seriously in both, with the foresight to overhaul their chemistry and manufacturing platforms together.