Oligonucleotide Manufacturing: Scale Up of Oligonucleotide Synthesis

Oligonucleotides Demand

The demand for oligonucleotides to treat a range of diseases, many of which were previously not treated with the same effectiveness by traditional medicines, appears to be at the early stages of a sustained upward trend. Between 2019 and 2023, there have been 10 new oligonucleotides that entered the market and the production of existing oligonucleotides as well as the development of new treatments only seems to be increasing. This growth is evident with the forecasted value in the market going from US$5.2 billion in 2020 to US$26.1 billion by 2030.

Challenges in Current Upstream Synthesis Process with Oligonucleotides

The main challenges with the current upstream SPOS process that industry is experiencing are the following:

• Quantity of Product: The SPOS leads to batches of final product of less than 10 kilograms.
• Solid Supports in SPOS Synthesizer: There are limitations of solid support loading capacity, i.e., amount of product that can be synthesized per unit volume of support, which appears to be the main barrier to large scale synthesis production using SPOS.
• Large Volumes of Waste: Solvents, e.g., Acetonitrile, are used in large volumes at this stage with many washing steps in between the traditional deprotection, coupling, oxidation, and capping steps.
• Emission Limits: With the introduction of more stringent emission limits on sites, the introduction of an oligonucleotide synthesis with reduced solvent usage/waste would be welcome. Treating and preventing harmful and high loaded solvent vent streams at source remains a best practice on all sites.
• Costs: High costs associated with solvents, reagents, and specialized equipment
• Equipment: Specialized synthesis equipment usage, such as a synthesizer with hydraulic pistons required with associated controls
• Safety: A high number of solvents and reagents at numerous steps of SPOS makes the safe handling of this paramount.

SPOS Process Optimization

The SPOS optimization potential is certainly present with industry optimizing their current plants and process—first and foremost, in conjunction with researching various SPOS configurations. Some optimization methods and novel SPOS configurations that are being developed are:

• Solvent Choice: By trial-testing different solvents, it may result in less swelling of the packing, a greener more sustainable solvent may be available, or a different solvent may result in easier purification of the oligonucleotide downstream.
• Column Packing Medium: The trial of various packing mediums could result in a higher loading capacity, with manufacturers improving packing mediums continuously.
• Equipment Optimization: Optimizing the equipment itself, such as implementing dynamic axial compression (DAC) synthesizers, may reduce the void spaces in the columns.
• Solvent Recycling: Re-using solvents in washing steps is beneficial, but the unclear regulatory standpoint of this does not lead to industry implementing recycling in many cases.
• Fluidized Bed with the Solid Support in Motion: The change from packed beds to fluidized beds would result in better mixing of reagents with solid phase and prevent channeling experienced in packed beds.
• Multiple Vessel Approach: Several mobile column/synthesizers in operation e.g. 4 No. column/synthesizers, one for each step (e.g., deblocking, coupling, oxidation, capping) so each step can occur in parallel and prevent bottlenecking.
• Continuous Synthesizer: (See Figure 1 for schematic of process). This approach involves a new equipment which would house every SPOS step (patent pending). A cartridge which contains a non-woven fiber (new solid phase material) inside starts at the beginning of the conveyor belt. Four stations (deblocking, coupling, oxidation, capping) within the equipment are all fixed with feed and recycle lines for solvent, reagents, etc. and all have an associated washing station. The cartridge starts at station one with a preloaded amidite and is moved using a conveyor belt to each station. At the end of the process, the cartridge contains an oligonucleotide chain on the non-woven support ready for cleavage and deprotection. (Arrowhead Pharmaceuticals, TIDES25)

Figure 1: Continuous Synthesizer

Potential Upstream Oligonucleotide Synthesis Techniques

The SPOS process optimization methods described above may improve efficiency and meet demand in the near future. However, to anticipate and adequately prepare for the growing demand of oligonucleotides, alternative methods may need to be developed at a large industry scale. The methods highlighted below rely on Liquid Phase Oligonucleotide Synthesis (LPOS) as the industry standard in the future:

Chemical Method

This method differs from traditional SPOS by removing the solid support entirely. The starting amidite, i.e., building block, must be deprotected, which is typically done using an acid the same as traditional SPOS but in a solution. The coupling process then follows, which involves reacting amidites in solution by using an activator, e.g., Tetrazole, which extends the oligonucleotide chain. A quenching agent is added to stop the reaction. An acid is then added to ensure that the chain is oxidized and stable. This process is repeated until the desired number of bases in the oligonucleotide are achieved.

After each step, i.e., deprotection, coupling, oxidation, the solution requires purification (i.e., chromatography, precipitation, etc.) to remove impurities, reagents, acids, quenching agents, activators, etc. This method could be completed in a traditional reactor or plug flow reactor in conjunction with the desired purification process in between each step.

Figure 2: Chemical Method Process Schematic

Liquid, Soluble Support Method

This method differs from traditional SPOS by replacing the solid support with a liquid, soluble support. It uses a liquid, soluble support, for example, polyethylene glycol (PEG) or cellulose acetate, so that the amidite can be attached. The benefit of using a large molecular liquid support is the ease of purifying between synthesis steps (i.e., this is the main benefit, compared to the chemical method).

The soluble support with the first amidite in solution is contained within the reactor (batch case) it is then deprotected with the addition of an acid, the solution is then pumped through a purification step which returns the larger molecular oligonucleotide to the reactor and the smaller molecular acid is sent to waste. With the oligonucleotide chain now deprotected, the amidite in solution is added to the reactor to add to the chain using an activator and quenching agent to start and stop reaction, again this is pumped through a purifying step, returning the oligonucleotide to the reactor. An acid is added to the reactor to ensure the chain is oxidized and stable. This process is repeated until the desired number of bases in the oligonucleotide are achieved. The solution then goes through a cleavage process to remove the soluble support and isolating the oligonucleotide.

This purifying step could be completed using an Organic Solvent Nanofiltration (OSN), which uses a membrane to separate molecules based on their molecular weight, hence the benefits of the soluble support. The process steps for chemical method are followed as they are presented above, but the purifying step is OSN at every step.

This method also removes the necessity for a complex synthesizer/column equipment and moves to a traditional reactor (batch) or plug flow reactor (continuous) with the nanofiltration equipment used in conjunction to enable the oligonucleotide synthesis.

A number of starting materials and soluble supports have been experimented with using this method, including traditional Phosphonamidite Method, H-Phosphonate Method, Triester Method, and using an Ionic liquid support. Ionic liquid supported synthesis (ILSS) papers have showed that its loading capacity of 2-12mmolg-1 which has benefits compared to other soluble supports with <0.2mmolg-1 and solid supports as low as 0.1mmolg-1.

Figure 3: Soluble Support Method Process Schematic

Enzymatic

This method uses enzymes to react amidites to the growing oligonucleotide chain by starting with a catalytic template in a pot/reactor. The coupling process involves reacting amidites in solution by using an enzyme which extends the oligonucleotide chain on the catalytic template to its targeted size.

This solution can then be purified so that any unreacted amidites or enzymes are removed from the solution. The oligonucleotide chain attached to the catalytic template then needs to be cleaved; this is achieved by adding an endonuclease. Again, a purifying step to remove endonuclease from the final product is required. This method can be completed in a reactor, with the enzyme being added to react the amidites to the chain and the endonuclease to cleave the oligonucleotide from the template.

There are two different techniques applicable to this method:

1. Template-Independent: Utilizes enzyme and doesn’t require a pre-synthesized template. The natural extension is uncontrolled and not very efficient.
2. Template-Dependant: Needs a pre-synthesized catalytic template and utilizes enzymes, which extend the sequences. This technique is more efficient and more controlled.

Figure 4: Enzymatic Method Process Schematic

Convergent Liquid Phase Synthesis

This method combines fragments of oligonucleotide chains previously synthesized from SPOS by using either a chemical method (batch or continuous, as described above), liquid soluble method (batch or continuous, as described above), or enzymatic method (batch, as described above).

Final Thoughts

Uncertainty within the current regulatory landscape may be contributing to slower adoption or causing hesitation among companies to implement novel technologies. This is particularly evident given that the forthcoming regulatory standards being issued in 2026 make no mention of liquid phase processing. However, in light of increasing demand for innovative oligonucleotide-based therapies, it is not unforeseeable that a method as listed above or another novel technology or method may become the industry standard for oligonucleotide synthesis.