Important Processing Steps and Considerations in Oligonucleotide Manufacturing

The oligonucleotide industry is experiencing a surge of interest and momentum thanks to the discovery of the CRISPR-Cas9 system and the development of RNA-based vaccines and therapies. Oligonucleotides represent one of the most promising therapeutic modalities for the foreseeable future as evidenced by over 300 candidates in clinical trials. Industry insiders predict that roughly 10% of new drug approvals by the FDA will be oligonucleotide-based, which emphasizes the need to address some key challenges with how oligonucleotide manufacturing can be effectively and reproducibly scaled topping the list.

Asahi Kasei Bioprocess, an industry leading equipment provider to oligonucleotide developers around the globe, has published a new infographic highlighting The Fundamentals of Oligonucleotide Manufacturing. The infographic examines the industry outlook and key trends for oligonucleotide manufacturing along with the goals and strategy to accomplish key upstream manufacturing steps. Solid phase synthesis, which remains the gold standard for manufacturing, cleavage and deprotection, and purification systems and columns are covered along with important considerations to achieve scalable production.

Solid phase Oligosynthesis

For mid- to large-scale oligonucleotide synthesis the flow-through synthesis column serves as solid support during the synthesis process and ensures an equal distribution of phosphoramidites and reagents to all beads in the packed bed. As a chemical process involving solvents like acetonitrile (ACN) and toluene, the synthesis column needs to accommodate fluctuations in column volume resulting from solvent interactions. It is also important to ensure all bead surfaces of the solid support are contacted by amidite and reagent in order to obtain the highest yield of crude oligonucleotide during synthesis. For scale up to larger column diameters, robust column flow distribution becomes an important driver for yield.

Cleavage & Deprotection

After successful synthesis, cleavage and deprotection separates the crude oligo from the solid support and removes the protecting group from the now dissolved oligos. These processes require harsh chemicals and elevated temperatures (in the case of RNA). Fully automated software-controlled systems to manage both cleavage and deprotection validated for mid- to large-scale GMP manufacturing can expedite and standardize this stage in oligo production.


There is typically a choice in chromatographic purification between reversed phase (RP) purification and ion exchange (IEX) chromatography each with its own advantages and disadvantages. Whether purifying in RP mode, IEX mode, or both, the mechanical design of the MPLC or HPLC System is crucial where the safety rating and sanitary design are important considerations. The purification system must also be capable of delivering consistent gradients for effective shortmer and longmer impurity removal while maintaining high oligo recovery. For commercial production, the system will also, ideally, offer user-friendly control software that can connect to the plantwide automation platform.

With respect to purification column, mid-sized oligonucleotides favor the use of spherical chromatography media with relatively small pore size, best packed with dynamic axial compression (DAC), where bulk media is slurried, transferred into the empty column and compressed into a packed bed by an axial piston. Therefore, the columns must withstand backpressures of 10-50 bar, depending on bed height, temperature, eluent conditions, and flow rate. Using a reproducible packing platform over manual processes allows for more consistent packing and reduces any preferential settling of large particles in the column prior to compression. Of course, column packing should be qualified using one or more column performance tests to ensure successful packing of the bulk media into the DAC column.

Asahi Kasei Bioprocess offers novel equipment from columns, systems and automation solutions to span key unit operations of oligonucleotide manufacturing from synthesis through purification.

To download the infographic, please visit: The Fundamentals of Oligonucleotide Manufacturing.

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