Oligonucleotide developers face a challenge. Traditional manufacturing methods work in the lab, however, they use toxic solvents and require extensive processing which makes them hard to scale up.

Phosphoramidite chemistry is the most widely used production method. The approach is based on the sequential addition of nucleosides to form an oligonucleotide chain. While effective, it is not efficient, according to Kevin Norrett, COO at protein engineering company Codexis.

“Traditional phosphoramidite chemistry processes for a 20-nucleotide oligo is a consecutive 160-step chemical process that requires large-scale management of acetonitrile and other toxic solvents along with significant downstream purification to produce siRNA therapeutics. The chemical nature of this process produces a large number of side reactions, or impurities, that are hazardous to a patient, which is the reason significant purification is required.

“To put this large-scale solvent use in perspective, 1,000 kg of acetonitrile is needed to produce 1 kg of siRNA. The ability for phosphoramidite chemistry processes to scale to meet the coming siRNA therapeutic demand in a safe, sustainable, and economical way is questionable, at best.”

Furthermore, the reactor technologies and solvent storage systems needed for phosphoramidite chemistry processes are expensive.

Norrett tells GEN: “The facilities required to handle the large volumes of organic solvents, stainless steel columns, and purification resins, are expensive and require massive infrastructure investments to only meet a portion of the anticipated demand of siRNA.

“With more than 60% of investigational siRNA compounds representing large-volume drugs, there is a dire need for alternative methods to compensate for the pending influx of therapeutics,” he says.

Market expectations

At present, only six siRNA therapeutics are approved. However, more are expected over the next decade as there are 450 investigational siRNA compounds in development, some 40 of which are already in late-stage clinical trials.

And this likely market growth is another reason why the industry needs to rethink production, according to Norrett.

“This translates to tens of thousands of kilograms of RNA demand and as a result, a new process is required that can support the scale of not only the number of compounds to be approved, but also drug volume for the patient populations for disease states being investigated.”

With this in mind, Codexis has developed an enzymatic oligonucleotide production platform that it claims bypasses the challenges associated with traditional methods.

The platform is a set of enzymatic tools designed to create full-length, therapeutically relevant RNA oligonucleotides. It uses two steps: a sequential nucleotide elongation cycle; and enzymatic ligation, which stitches together short strands of RNA. This compares favorably with traditional phosphoramidite chemistry-based approaches, which employ eight chemical steps for each nucleotide addition.

Codexis’ system can also add targeting agents designed to ensure delivery to the right tissues, which is a key feature, Norrett says.

“There are over 450 products in development right now for a variety of cardiovascular, central nervous system, and oncology diseases, with the vast majority of them using some type of tissue targeting agent. The entire process, core technology elongation and enzymatic ligation, is completed in an aqueous solution as opposed to using toxic organic solvents, such as acetonitrile. As a result, we are able to avoid the cost of managing the waste streams for metric tons of organic solvents, and don’t need to build facilities that incorporate fire suppression and explosion-proof construction designs for our manufacturing process.”

He suggested the platform “can enable drug developers to enter a space that would otherwise be inaccessible from a capital and scale standpoint and enable the growth of this important class of emerging therapeutics.

“As a result, we are seeing a lot of commercial interest, particularly from large pharma and their contract manufacturing organizations that are in need of an alternative to traditional chemistry to address the scale limitations of existing methods, and to do so in a sustainable way.”

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