Key takeaways

• The "solid" ceiling: Traditional solid-phase oligonucleotide synthesis (SPOS) works well for R&D but struggles with the metric-ton scale required for blockbuster drugs like Inclisiran, primarily due to massive solvent waste and reagent excess.
• Liquid phase returns: Technologies like AJIPHASE® are reviving liquid-phase synthesis (LPOS) by using soluble anchors, allowing oligos to be manufactured in standard chemical reactors with significantly improved atom economy.
• Enzymes enter the chat: Template-independent enzymatic synthesis (TdT) promises to eliminate organic solvents entirely, offering a "green" path to longer, cleaner DNA and RNA constructs that chemical methods cannot match.
• Continuous purification: Moving from batch chromatography to continuous processes (like MCSGP) is proving critical for reducing the "Process Mass Intensity" (PMI) and cost of goods for commercial RNA drugs.

For thirty years, oligonucleotide synthesis was a niche sport. It was the domain of rare diseases—Duchenne muscular dystrophy, spinal muscular atrophy—where patient populations were small and manufacturing a few kilograms of Active Pharmaceutical Ingredient (API) per year was sufficient.

That era ended with the approval of Leqvio (inclisiran). Suddenly, the industry faced a therapy targeting hypercholesterolemia—a condition affecting millions, not hundreds. This shift from "orphan" to "mainstream" has triggered a manufacturing crisis. The traditional solid-phase methods that built the industry are now hitting a physical and environmental wall. To meet the demand for tons of RNA, manufacturers are rewriting the rulebook, looking backward to liquid-phase chemistry and forward to biology-driven enzymatic assembly.

The waste problem: Why solid-phase struggles at scale

The gold standard for decades has been solid-phase oligonucleotide synthesis (SPOS). It is reliable, automatable, and perfect for the milligram scales needed for research. But SPOS is inherently wasteful. It requires a massive excess of reagents to drive reactions to completion on a resin bead, and extensive washing steps between every cycle.

The metric that haunts manufacturers is "Process Mass Intensity" (PMI)—the mass of raw materials used to make one kilogram of drug. For small molecules, a PMI of 100 is typical. For oligonucleotides, PMIs often exceed 4,000 [1]. Scaling SPOS to produce metric tons of siRNA results in lakes of hazardous organic waste (like acetonitrile), creating a sustainability nightmare that regulators and environmental mandates are no longer willing to ignore.

Back to the future: Liquid-phase synthesis (LPOS)

To solve the scale problem, chemists are dusting off an old concept: doing chemistry in solution. Liquid-phase oligonucleotide synthesis (LPOS), pioneered by technologies like Ajinomoto’s AJIPHASE®, uses a soluble anchor molecule instead of a solid resin bead.

This hybrid approach offers a decisive advantage for commercial manufacturing: it uses standard industrial reactors. You don't need specialized, capital-intensive solid-phase synthesizers. By performing the reaction in a homogeneous solution, mixing is more efficient, less reagent is required, and the "anchor" allows the growing oligo chain to be precipitated and filtered easily between steps. This can cut solvent consumption by up to 60%, making the "ton-scale" production of ASOs and siRNAs economically viable for the first time [2].

The enzymatic frontier: Writing DNA with biology

While LPOS refines chemical synthesis, enzymatic synthesis reimagines it entirely. This method uses Terminal Deoxynucleotidyl Transferase (TdT), an enzyme that adds nucleotides to a DNA strand without a template.

The promise of enzymatic oligonucleotide synthesis is the elimination of the harsh organic solvents required by phosphoramidite chemistry. It takes place in aqueous conditions—water. While currently slower and more expensive than chemical methods for short sequences, enzymatic synthesis excels at producing ultra-long strands (>200 mers) that chemical methods cannot touch due to accumulating error rates. As companies like DNA Script and Ansa Biotechnologies mature their platforms, this "green" manufacturing method is poised to dominate the production of long guide RNAs for CRISPR and synthetic genes [3].

Continuous purification: The final bottleneck

Synthesizing the molecule is only half the battle; purifying it is the other. Traditional batch chromatography is the bottleneck of the downstream process, consuming vast amounts of solvent and time.

Innovations in continuous chromatography, specifically Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), are transforming this step. By continuously recycling the "impure" side fractions that would normally be discarded in a batch process, MCSGP increases yield by 30-60% while simultaneously reducing solvent use. For a high-cost therapeutic, recovering that extra 30% of product can mean the difference between a profitable drug and a commercial failure [4].

Conclusion: The industrial revolution of RNA

We are witnessing the industrial revolution of nucleic acids. Just as the textile industry moved from hand looms to factories, oligonucleotide synthesis is moving from benchtop synthesizers to industrial reactors. By integrating liquid-phase scalability, enzymatic precision, and continuous processing, the industry is finally building the infrastructure capable of delivering genetic medicine to the world—not just the few.

References

1. Andrews, B. I., et al. (2021). Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing. The Journal of Organic Chemistry, 86(1), 49–61.

2. Takahashi, D., et al. (2017). AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy. Angewandte Chemie International Edition, 56(27), 7803–7807.

3. Eisenstein, M. (2020). Enzymatic DNA synthesis enters new phase. Nature Biotechnology, 38, 1113–1115.

4. Müller-Späth, T., et al. (2021). Increasing Yield and Purity in the Downstream Processing of Oligonucleotides. Bachem Insights.

5. Research and Markets. (2024). Oligonucleotide Synthesis Market Size, Share & Trends Analysis Report. Market Industry Report.