Dr. Janik Kaufmann, technical sales specialist Oligonucleotides, Hongene Biotech, looks at why chemoenzymatic technologies are moving into the spotlight.
The oligonucleotide therapeutics industry is entering a new phase of growth. Following the clinical and commercial success of the first wave of antisense oligonucleotides (ASOs), siRNAs, and emerging gene-editing modalities primarily addressing rare diseases, market projections suggest that several upcoming siRNA therapeutics targeting highly prevalent diseases such as cardiovascular, metabolic, and liver diseases could reach patient populations far beyond the rare disease indications. As a result, manufacturing demand is expected to increase substantially over the coming decade making production capacity, scalability, and cost of goods decisive factors for commercial success.
Meeting this demand using conventional manufacturing methods alone would prove challenging. While the pharmaceutical industry has relied on phosphoramidite-based solid-phase oligonucleotide synthesis (SPOS) for more than 40 years, the growing scale requirements of future RNA medicines are prompting manufacturers to evaluate alternative approaches. Currently, there are several alternative phosphoramidite-based technologies under investigation, such as SPOS in stirred-bed reactors, liquid-phase oligonucleotide synthesis (LPOS) options that use hydrophobic supports or reactors with selectively permeable membranes, and mechanochemistry approaches. Among the most attractive developments are fully enzymatic technologies that rely on nucleoside triphosphate (NTP)-based instead of phosphoramidite-based raw materials, which eventually promise improved yield, purity, scalability, sustainability, and reduced cost of goods.
However, such enzymatic synthesis approaches are not yet ready for large-scale manufacturing. Supply of NTP raw materials needs to be established, and enzymes require engineering to incorporate chemically modified NTPs into oligonucleotide products with sufficient high fidelity and yield. Enzymatic processes have been successfully scaled up to yield gram quantities of siRNA, but the multi-kg manufacturing scales realised by SPOS today have not yet been demonstrated.
In this context, SPOS could be seen as the first generation of oligonucleotide manufacturing technology, while fully enzymatic processes represent a potential third generation. A hybrid approach called chemoenzymatic ligation bridges the well-established, well-characterised SPOS process and the future enzymatic processes. This second generation uses oligonucleotide fragments currently synthesised by SPOS, often referred to as blockmers, that are assembled by enzymatic ligation into the final full-length product. The attractiveness of chemoenzymatic manufacturing lies in its ability to retain the flexibility of established SPOS chemistry while addressing several of its inherent limitations.
One major drawback of SPOS is that overall yield decreases with every additional coupling cycle. Although phosphoramidite couplings proceed with very high conversion, residual shortmer impurities – truncated sequences where the last monomer addition was unsuccessful – are inevitable. Shortmer impurities are chemically capped before the coupling cycle of the next monomer begins, rendering them unreactive and therefore preventing their elongation during the remaining cycles. However, these shortmer impurities persist in the final crude product. Besides shortmers, additional impurities can form in side-reactions occurring during other SPOS reaction steps. Many of these impurities are structurally closely related to the full-length product, making them difficult to separate and creating additional challenges during purification. This challenge becomes particularly pronounced for long modalities, such as single guide RNAs (sgRNAs) used in gene-editing applications.
Chemoenzymatic ligation addresses these limitations by distributing the manufacturing burden from repeated chemical coupling cycles on one long molecule to fewer cycles on multiple smaller fragments. Fewer repetitive chemical synthesis cycles reduce the accumulation of synthesis-related impurities which results in high-purity blockmers. The enzymatic ligation reaction itself typically does not generate new impurities and due to the difference in size between blockmers and the much longer ligated full-length product, non-ligated blockmers and their shortmer impurities can be efficiently purged during purification. Furthermore, blockmer yields are higher than for long sequences synthesised by SPOS, and solid supports with very high loadings can be utilized to achieve higher scales with the same synthesis columns reducing the cost of manufacturing and environmental footprint. Subsequent enzymatic ligation of blockmers can be performed at large scale in simple batch reactors or single-use bioreactors, which are standard equipment for scalable manufacturing.
Importantly, this technology is no longer a theoretical concept. It has been demonstrated that chemoenzymatic ligation can be successfully applied to manufacture cGMP therapeutic siRNA products advancing into clinical development. Historically, oligonucleotide manufacturing decisions were driven primarily by quality and regulatory compliance. With the projected increase in manufacturing scale, sustainability is becoming an equally important consideration. SPOS is highly resource intensive,
requiring large quantities of reagents, solvents, and consumables. High volumes of acetonitrile are required to wash the solid support after each cycle step and for a simple single-stranded 21mer, a minimum of 80 steps is already required. An average process mass intensity (PMI) of 4299 has been calculated for typical SPOS processes, corresponding to an average PMI of 199 per nucleotide. By comparison, median PMI values for traditional small molecule drugs are in the range of 168 to 308 only.
Because blockmer yield is typically high and ligation reactions are performed in aqueous systems, chemoenzymatic approaches offer a meaningful opportunity to reduce this environmental burden. Blockmers can be purified by chromatography prior to enzymatic ligation. However, sufficiently pure crude blockmers can be successfully employed in the ligation reaction without prior purification, affording similar or even higher purities for the full-length product than can be achieved by conventional SPOS. Removal of the blockmer purification steps will further reduce PMI, manufacturing time and cost. An ideal process flow can be envisioned in which crude blockmers are enzymatically ligated, yielding a product of sufficient crude purity to be further processed as liquid active pharmaceutical ingredient (API) without the need for any purification or lyophilisation steps. Looking further ahead, third-generation enzymatic manufacturing technologies may eventually enable environmentally sustainable aqueous processes that are cost-competitive with SPOS and chemoenzymatic ligation.
The future of oligonucleotide manufacturing will most likely involve a combination of established chemical synthesis and enzymatic technologies. Nevertheless, the trend is becoming clear. Growing demand for siRNA, sgRNA and other advanced oligonucleotide therapeutics is driving the industry toward manufacturing platforms that offer better scalability, lower environmental impact and reduced cost of goods. Chemoenzymatic ligation has emerged as a promising solution because it can simultaneously improve yield, enhance purity, and reduce the environmental footprint. As the industry shifts from treating thousands of patients to potentially millions, manufacturing innovation will become increasingly important. The companies best positioned for the future of RNA medicines may well be those that successfully translate manufacturing technologies into scalable, sustainable and commercially viable production platforms.