The manufacturing process of individualized ASOs consists of well-studied phosphoramidite (base building blocks) chemistry, known since the 1960s. And is carried out with automated steps.
Elongation of the ASO strand takes place on a solid support, followed by cleavage and deprotection, chromatographic purification, detritylation, ultrafiltration/diafiltration (UF/DF), and concentration and/or lyophilization to arrive at a solution or powdered ingredient.

Solid-Phase Synthesis
Over the last few decades, solid-phase synthesis has been successfully applied for commercial manufacturing of ASOs. This is possible due to the simple and efficient reaction sequence leading to high quality of the produced nucleotides. Protected nucleoside building blocks are assembled into a pre-defined sequence through an automated chemical process. Cost of the solid support, relatively large consumption of expensive chemicals (GMP-grade starting materials and solvents), and more challenging scale-ups are the limiting factors.
During solid-phase reaction, synthesis steps are computer-controlled and fully automated. The reagents are pumped through the column (solid support) sequentially for the reactions. The nucleotide chain grows from 3′ to 5′ direction, and performance of a four-step reaction cycle enables elongation by one monomer:
- Deprotection: an acid is used to remove the protecting group on the 5′ end of the elongating chain.
- Conjugation: The 3′-OH ribose building block is activated by tetrazolium to form an active intermediate, which is then linked to the elongating oligonucleotide chain.
- Oxidation: The newly added nucleotide is attached to the oligonucleotide chain on the solid phase support by a phosphoester bond and then oxidized to stable pentavalent phosphorus.
- Capping: The 5′-OH of the oligonucleotide is treated with a capping reagent to prevent re-coupling.
This procedure, which includes solvent washes (acetonitrile) after each step, is repeated until the final protected oligonucleotide chain reaches its desired length. When PS linkage is required, a sulfurizing reagent is used in the reaction step. All reactions in the cycle are rapid: a single cycle takes approximately 45 min and a full synthesis of a 20-mer is complete in less than 24 h.
In-line reaction progress detection with UV and conductivity monitoring provides vital data to evaluate completion of each synthesis step. Although the coupling step nears 100% efficiency, some free 5′-OH groups are always present. To prevent nonreacted species to enter another cycle, which would result in n – 1 impurities, the free 5′-OH groups are capped with acetic anhydride.
The final step, cleavage and deprotection, takes place and involves release of the oligonucleotide chain from the solid support and deprotection of the remaining protecting group. Once the cleavage is done, the support is filtered from the recovered ASO solution, washed, and discarded and the filtrate is collected. To confirm the success of the synthesis, purity, identity and content are analyzed.
ASO Impurities
Synthesis provides crude material with a limited purity (60–80%, depending on the type and length of the product). The mixture consists of a full-length product accompanied by a mix of impurities. The longer the oligonucleotide chain, the greater the number of synthetic steps, the higher the impurity levels.
Impurities originating from starting materials can be divided into three categories: nonreactive, the least critical (removed with washes during synthesis), reactive, not critical (built in the ASO chain, but removed together with protecting groups without impacting quality of the synthesized product), and reactive, critical (stay in the ASO chain, forming a different product).
The platform nature of ASO synthesis permit the prediction of common product and process-related impurities, and facilitates the development and optimization of systematic purification steps to arrive at the required clinical quality material.
Purification of ASOs by Reversed-Phase (RP-HPLC) and Anion Exchange (AEX-HPLC) Chromatography
Chromatic purification technologies have been used to purify oligonucleotides since the 1970s. RP-HPLC employs a hydrophobic stationary phase to retain the oligonucleotide via its hydrophobic 5′-DMT group. Impurities without the DMT-group are washed out. AEX-HPLC uses a quaternary ammonium-functionalized stationary phase, which binds the negatively charged oligonucleotide at low salt concentrations. Higher number of charges results in increased retention compared to that of lower charged species. Hence able to differentiate impurities differing by one chain length (shortmers and longmers, impurities in phosphorothioated oligonucleotides, and the separation of diastereomers of phosphorothioated oligonucleotides.
Ultrafiltration
Ultrafiltration is a commonly utilized step in the manufacturing of therapeutic ASOs, with well-established industry standards for process development and scale up. It uses a set of pumps and tubing to circulate the crude mix through a porous membrane with the determined molecular weight cutoff, where small molecules such as water, solvents, inorganic and organic molecules, and byproducts pass through the membrane, whereas the much larger oligonucleotides are diverted back.
Lyophilization
The last step in downstream processing is the isolation of powder API, giving a low-density, hygroscopic, electrostatic, amorphous solid. First, the ASO solution is frozen, and sublimation is induced with applied vacuum and gradually delivered heat. Primary drying is complete when the temperatures of the shelf and the product becomes equal. Throughout this step, most of the water sublimates; however, some water remains adsorbed and is eliminated with stronger vacuum and further heating. The lyophilization step might take up to 1 week to complete, is energy and manpower intensive. Powdered API shows superior chemical and microbial stability compared to the solution API.