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ASO & siRNA: One Category, Two Manufacturing Problems
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Nusinersen, zilganersen, and vutrisiran all fall under the broad category of oligonucleotide therapeutics. But beyond that, they have little in common. Nusinersen and zilganersen are administered intrathecally, while vutrisiran is injected under the skin to target hepatocytes.1-3
These structural and delivery differences translate into distinct manufacturing requirements. In this article, we’ll explore how these therapies illustrate the diversity of oligonucleotide manufacturing and why treating them as a single category ignores crucial production differences.
Two architectures of oligonucleotide therapeutics
Two major classes of oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and small interfering RNA (siRNA), perfectly illustrate how molecular design dictates manufacturing requirements.
The main difference concerns their strand architecture:
- An ASO is a single strand, typically 18 to 30 nucleotides, featuring chemical modifications that improve nuclease resistance and fine-tune binding behavior.4-5 Nusinersen, for example, is an 18-mer ASO built with a phosphorothioate backbone and 2′-O-methoxyethyl (2′-MOE) modifications.1
- By contrast, an siRNA requires two complementary strands: the guide (antisense) and the passenger (sense), annealed to form a duplex.6 Vutrisiran is one such chemically modified double-stranded siRNA, utilizing a conjugated tri-GalNAc ligand to target liver cells.3
While both modalities rely on chemical modifications to control stability, binding affinity, and disposition, their specific design rules differ significantly. Phosphorothioate linkages can improve nuclease resistance while also affecting protein binding and distribution.7 Sugar modifications, such as 2′-MOE, 2′-O-methyl, and 2′-fluoro, can further influence affinity and biological activity.8
Ultimately, the drug's intended mechanism governs exactly how these modifications are deployed. In an siRNA, the chemistry must be carefully balanced to preserve the guide strand’s ability to direct the RNA-induced silencing complex (RISC) to its target.6 In a splice-switching ASO, the molecule must successfully bind its target RNA to alter splicing without inadvertently triggering RNase H1-mediated cleavage.5
Chemical modification is thus a highly specialized step required to ensure the biological mechanism works.
Two mechanisms of action
While ASOs and siRNAs both rely on sequence-specific base pairing to locate their target RNA, their paths diverge after binding.
ASOs execute their function through several distinct mechanisms, depending on their chemical design:5
- Gapmers recruit RNase H1 to cleave the target RNA.
- Steric-blocking ASOs bind directly to the RNA, physically obstructing cellular machinery and interactions at that site.
- Splice-switching ASOs bind to pre-mRNA to alter how it is processed.
Nusinersen is a splice-switching ASO. It binds SMN2 pre-mRNA near exon 7 and promotes exon 7 inclusion, increasing full-length SMN protein production.1 Meanwhile, zilganersen (FDA-approved in September 20269) works differently. It binds GFAP pre-mRNA and promotes its degradation, reducing glial fibrillary acidic protein (GFAP) production.2
In contrast, siRNA essentially hijacks the cell’s natural RNA interference (RNAi) pathway. Once delivered inside the cell, the siRNA duplex separates. The passenger strand is discarded, while the guide strand is loaded into the RNA-induced silencing complex (RISC). There, the Argonaute 2 enzyme uses the guide strand to repeatedly recognize and cleave complementary target mRNA.10-11 Vutrisiran uses this same pathway to degrade transthyretin (TTR) mRNA, lowering serum TTR levels and clearing tissue deposits.3
Conceptually, the difference comes down to occupancy versus cleavage. Steric-blocking ASOs sit on their target, while siRNAs guide an enzyme to repeatedly cut it.11 The distinction is useful, but it does not determine clinical dose or dosing interval on its own. In practice, a drug's duration of effect depends on a complex interplay of tissue distribution, cellular uptake, target turnover, and the molecule’s bespoke chemical design.
Two delivery problems in oligonucleotide therapy
Oligonucleotide delivery is notoriously tissue-dependent. The current landscape is largely defined by two established strategies: physically bypassing the blood–brain barrier to access the central nervous system (CNS), and utilizing targeted conjugation to shuttle payloads directly to the liver. Naturally, each approach imposes entirely different requirements on drug product design and clinical execution.
- Reaching the central nervous system
Large, negatively charged oligonucleotides cannot easily cross an intact blood–brain barrier when administered systemically.12 Therapies like nusinersen and zilganersen circumvent this obstacle via direct intrathecal administration.
While this route successfully delivers the drug to the CNS, it introduces significant clinical friction. Patients face repeated lumbar punctures, requiring specialized clinicians and dedicated procedural capacity. Furthermore, anatomical complications like scoliosis or spinal fusion can make administration difficult or even impossible for some patients.
- Reaching the liver
Vutrisiran employs a completely different strategy. It uses a conjugated tri-GalNAc ligand to bind the asialoglycoprotein receptor on hepatocytes, driving targeted cellular uptake following a simple subcutaneous injection.3
GalNAc conjugation has revolutionized the field, turning reliable hepatocyte targeting into a clinical standard.14 However, it does not solve the modality's broader delivery problem of achieving effective, targeted uptake in tissues outside the liver.
Recent approvals, however, highlight the sheer dominance of this liver-directed strategy. Fitusiran and plozasiran (both approved in 2025) are subcutaneous GalNAc-conjugated siRNAs, while donidalorsen (2025) is a subcutaneous GalNAc-conjugated ASO.15 By contrast, the recently approved zilganersen (September 2026) relies on intrathecal delivery to reach the brain.2.9 Ultimately, these clinical precedents prove that both ASOs and siRNAs can successfully leverage liver-targeted conjugation, while intrathecal administration remains the proven, yet demanding, route for CNS activity.
One target, four medicines: Approved oligonucleotides against TTR
Four approved oligonucleotides against transthyretin (TTR) mRNA show how widely design can vary within a single target.
The contrast between inotersen and eplontersen is particularly revealing. Both are subcutaneous ASOs directed against TTR. However, eplontersen utilizes a conjugated GalNAc ligand to actively promote hepatocyte uptake. From the manufacturing perspective, this design choice demands an additional conjugation step, complete with complex process and analytical controls that inotersen simply does not require.
Consequently, one cannot infer an oligonucleotide's true manufacturing implications from its class name alone. They are intrinsically tied to the molecule’s architecture, bespoke chemical modifications, conjugation or formulation strategy, and the specific quality attributes that must be rigorously controlled.
What changes in oligonucleotide manufacturing
The therapeutic differences between ASOs and siRNAs are well documented in the literature. Far less often discussed is what the biological and structural differences mean for manufacturing these active substances.
What a single strand already demands
An ASO is a single chain, making it the simpler of the two architectures. But it is by no means a simple product to manufacture.
Solid-phase synthesis builds the oligonucleotide chain one nucleotide at a time, and coupling efficiency is never a perfect 100% at each cycle. As a result, the crude material contains the full-length product alongside truncated sequences and other process-related impurities. As we explored in our previous article, these closely related species cannot be easily separated using conventional extraction or crystallization. Instead, high-resolution chromatographic purification is mandatory to control the impurity profile.16
Chemical modifications add another layer of complexity. For example, a fully phosphorothioate-modified backbone introduces a stereocenter at every single linkage. This results in a complex population of diastereomers rather than one single, stereochemically defined molecule.14
Finally, the route of administration affects downstream requirements. Intrathecal products, for instance, must meet exceptionally stringent endotoxin limits and other quality standards required for administration into the cerebrospinal fluid.14 For an ASO like nusinersen, downstream processing involves tangential flow filtration before the drug substance is formulated and lyophilized.
If single-stranded ASO is already a highly control-intensive product, let’s see what a duplex adds to that baseline.
A duplex is not twice a single strand
A therapeutic siRNA duplex is a two-component active substance whose quality depends on the identity and purity of each strand, as well as on the formation and integrity of the final duplex.6,17
This creates five practical manufacturing considerations:
1. Two separate syntheses
Both strands are produced via solid-phase phosphoramidite synthesis, utilizing the same cycle of deprotection, coupling, oxidation, and capping used for ASOs. However, the guide and passenger strands have completely different sequences and often differ in length and chemical modifications. If the siRNA is conjugated, the ligand must be chemically attached to a designated strand.
As a result, each strand demands its own dedicated control strategy to verify its identity, content, and impurity profile. If the strands are purified separately, these quality attributes must be confirmed before the process can advance to annealing.14
2. Two separate purifications, or perhaps just one?
The traditional approach is to purify and characterize each strand separately before annealing them into a duplex under controlled conditions.18
However, a viable alternative is to anneal the crude strands immediately after synthesis and purify the resulting duplex directly. Published data show this can sometimes yield a higher proportion of the target duplex while maintaining comparable overall purity.17-18 However, the most optimal route depends on the specific molecule, its impurity profile, and overall process performance.
3. Conjugation, where applicable
For a GalNAc-conjugated siRNA, the way the targeting ligand is incorporated is a process design decision with implications for manufacturing and control.14
- Post-synthesis conjugation: The ligand is attached via a separate chemical reaction after the oligonucleotide is synthesized. This route demands robust downstream controls to measure conjugation efficiency, residual unconjugated material, related impurities, and the stability of the linkage itself.
- Solid-support incorporation: The ligand is pre-loaded onto the solid support, and the oligonucleotide chain is assembled directly on top of it. This removes the separate conjugation step but turns the functionalized support into a critical raw material. The control burden shifts upstream to incoming material specifications (such as ligand loading and consistency), though the final active substance still requires full purity and identity testing.
Because these two strategies place completely different demands on process development and analytical control, the decision must be made early as it dictates whether the primary control burden rests on managing a downstream chemical reaction or qualifying an upstream raw material.
4. Annealing and duplex-related impurities
During annealing, the complementary strands combine at a highly controlled ratio and are subjected to specific thermal conditions to drive duplex formation. The goal is to consistently generate the target duplex while minimizing residual single strands and other process-related impurities, which can include:17
- Unpaired strands: Excess guide or passenger strands left over from an imprecise mixing ratio or incomplete annealing.
- Non-optimal duplexes: Formations containing strands with synthesis-driven impurities, such as truncated sequences or backbone variants.
- Annealing-related side products: Unintended chemical species (such as 2′,3′-isomers) that can form under specific process conditions.
Process parameters such as temperature, duration, concentration, and buffer composition affect impurity formation and must be strictly controlled.6,17
5. Analytical methods that assess both strands and the duplex
One cannot determine an siRNA's quality by analyzing the individual strands in isolation. Because an siRNA consists of two different strands, a broader range of peaks may appear than with a single-stranded ASO.17
For double-stranded oligonucleotides, purity should be tested with both a non-denaturing method, to allow measurement of single-strand residues, and a denaturing method. Reference standards are expected for the sense strand, the antisense strand, and the active substance itself.14
Analytical conditions are critical because duplex stability can fluctuate based on concentration and buffer composition.17 Methods must thus be meticulously designed and qualified to ensure reproducible results.
Moving from an ASO to an siRNA is not as simple as adding a second strand. The main difference is the additional rigorous controls needed to verify the final active substance. For a single-stranded ASO, purity testing focuses entirely on the intended chain and its process-related impurities. For an siRNA, testing must evaluate both strands individually and confirm they have formed the intended duplex, while precisely quantifying residual single strands and other impurities.
Two platforms, not one extended: How Polpharma API approaches oligonucleotide manufacturing
As we’ve established, single-stranded ASOs and double-stranded siRNAs share a chemical foundation, but the processes required to produce, purify, and control them differ considerably.
Polpharma API’s oligonucleotide capabilities cover both modalities. Our single-stranded ASO experience is built on products like nusinersen sodium, while our double-stranded siRNA platform includes vutrisiran sodium.
This separation is a practical necessity. Manufacturing a duplex requires distinct capabilities: strand-specific synthesis controls, precise annealing operations, specialized conjugation chemistry, and dual-level analytical methods that evaluate both the individual strands and the intact duplex. Rather than treating siRNA as an extension of single-stranded production, we operate distinct, validated capabilities tailored specifically to each platform.
One chemistry, two paths forward for oligonucleotide development
The oligonucleotide field is not moving toward a single, unified architecture. Instead, ASOs and siRNAs are evolving along parallel paths, driven largely by delivery realities. Liver-targeted conjugates have established a highly reliable route for both modalities, while intrathecal administration remains a critical approach for CNS therapies. Achieving targeted delivery beyond the liver remains the industry's next great frontier.
For contract manufacturers, building oligonucleotide therapeutics means going far beyond the shared synthetic chemistry. Every structural variable, from strand architecture and chemical modification to conjugation and analytical control, reshapes how the active substance is made and tested.
When a pipeline expands from a single-stranded ASO to a GalNAc-conjugated siRNA duplex, manufacturers cannot simply bolt new steps onto an existing process. They must ask which manufacturing, purification, and analytical controls to reconsider from the ground up.
Author
Senior Technologist
Technology Development Department
Polpharma API BU
Sources:
- U.S. Food and Drug Administration. SPINRAZA (nusinersen) injection, for intrathecal use — Prescribing Information. Biogen.
https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/209531lbl.pdf - U.S. Food and Drug Administration. ZANVASTRO (zilganersen sodium) injection, for intrathecal use — Prescribing Information, NDA 220210. Ionis Pharmaceuticals, 2026.
https://www.accessdata.fda.gov/drugsatfda_docs/label/2026/220210Orig1s000lbl.pdf - U.S. Food and Drug Administration. AMVUTTRA (vutrisiran) injection, for subcutaneous
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- Crooke, S.T., Vickers, T.A. & Liang, X. Phosphorothioate modified oligonucleotide–protein interactions. Nucleic Acids Research 48(10), 2020, 5235–5253.
https://academic.oup.com/nar/article/48/10/5235/5827665 - Liang, X. et al. Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides. Nucleic Acids Research, 2017.
https://pubmed.ncbi.nlm.nih.gov/28977508/ - U.S. Food and Drug Administration. FDA Approves First Drug to Treat Alexander Disease, press announcement, 2026.
https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-treat-alexander-disease - Matranga, C. et al. Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes. Cell, 2005.
https://www.cell.com/fulltext/S0092-8674(05)00922-0 - Deerberg, A. et al. Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 protein. PNAS, 2013.
https://www.pnas.org/doi/10.1073/pnas.1217838110 - Zanjko, L.C. et al. Safety and toxicity assessment of antisense oligonucleotides in brain-relevant human cellular models, 2026.
https://pmc.ncbi.nlm.nih.gov/articles/PMC13560553/ - Ionis Pharmaceuticals. ZANVASTRO (zilganersen) approved by the FDA as the first and only disease modifying treatment for Alexander disease, 3 September 2026. https://ir.ionis.com/news-releases/news-release-details/zanvastrotm-zilganersen-approved-fda-first-and-only-disease
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https://www.ema.europa.eu/en/documents/scientific-guideline/draft-guideline-development-manufacture-oligonucleotides_en.pdf - Cronin, J. & Ai-MIng, Y. Small RNA or oligonucleotide drugs and challenges in evaluating drug-drug interactions. Frontiers in Pharmacology, 2026.
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