SAFC has more than 25 years experience in the manufacturing of DNA and RNA synthesis reagents. Its main oligonucleotide reagent manufacturing facility is located in Hamburg, Germany. Annual amidite production capacity at the Hamburg plant is in excess of five tons, making it the largest such facility in the world, according to the company. Large-scale production facilities at the plant include 1,000 L glass-lined reactors in addition to mid-size reactors (20–200 L).
“The supply chain for phosphoramidites starts with plant-derived materials like corn starch, and encompasses both unprotected and protected nucleosides,” says Andreas Wolter, site manager at SAFC. “Whereas many unprotected nucleosides are manufactured in large scale for the production of nucleoside-based APIs like zidovudine or as food additives, other nucleosides can be made from these major raw material streams through simple chemical or biocatalytic transformations,” he adds.
SAFC taps into this established supply chain to obtain unprotected and protected nucleosides, which are converted to phosphoramidites in the Hamburg plant. “In contrast to other manufacturers, we employ large-scale chromatographic purification as the key step for the purification of our amidites,” says Wolter.
“This results in greatly increased consistency, and reduced lot-to-lot variations,” he adds. SAFC utilizes a multivendor strategy to ensure a stable supply of raw materials into the manufacturing pipeline. “While some vendors have dropped out of the market over time and others have entered it, we have not experienced any shortage in these materials over the past decade,” says Wolter.
In solid-phase synthesis of oligonucleotides, liquid reagents are used in each step of the synthesis cycle. Overall synthesis performance, and therefore total product yield and purity of the crude oligonucleotide, is highly dependent on the chemical purity of the monomers and the supporting liquid reagents.
Over the past decade or so EMD Millipore has been involved in liquid reagent supply for oligonucleotide synthesis. John Koterba, product manager solvents, highlights the commercial-scale logistics associated with scale-up from clinical volumes to commercial scale and how this impacts the supply chain and the impact to the customer/vendor relationship.
“Manufacturing gram to kilogram to multiple kilograms of APIs on an annual basis has an impact on the critical liquid reagents supply,” says Koterba. “EMD Millipore has made recent capacity enhancements to account for these commercial-scale requirements,” he adds.
Deblocking is a critical step in oligonucleotide synthesis, in which the protecting group from the 5´ hydroxyl moiety of nucleotides already incorporated into the growing nucleic acid are removed prior to the addition of the next phosphoramidite. Removal of the blocking group allows the unprotected 5´ hydroxyl moiety to react with a new phosphoramidite in a subsequent extension reaction.
“We can produce halogenated acid deblocking solutions in the range of 400–7,400 liters,” says Koterba. During the oligonucleotide synthesis cycle, typically 1–2% of oligonucleotide chains will contain unreacted 5´-hydroxyl groups that did not react with the phosphoramidite moiety.
These unreacted groups must be blocked from further elongation in order to prevent the introduction of errors into the final oligo product. Capping with an acetyl group renders these 5´ hydroxyl groups unreactive for subsequent synthesis steps.
Other companies represented at the meeting are developing chemical strategies to streamline the oligonucleotide synthesis process in order to reduce costs. Kyeong Eun Jung, Ph.D., senior director of oligonucleotide research and development at ST Pharm Co. (formerly Samchully Pharmaceutical) cites blockmer technology as a promising approach to more efficient oligonucleotide synthesis.
Conventional oligonucleotide synthesis involves the sequential coupling of monomeric nucleoside phosphoramidites. In the blockmer synthetic strategy, an oligonucleotide analog is generated by sequential coupling of short protected oligomers or blocks (for example, a dinucleotide) on a solid support. Advantages of this approach include a smaller number of synthesis cycles required to prepare an oligonucleotide, saving time and reducing the amount of starting reagent required.
“During synthesis of a 21 mer anticancer siRNA targeting noxin-like UV-induced anti-apoptotic protein, we reduced the number of coupling steps from 20 to 10,” says Dr. Jung. Another advantage is increased purity. “We have found that incorporation of a single dimer into a 21 mer siRNA can result in a 10% to 20% increase in purity,” he adds.
STPharm is also investigating the feasibility of more efficient recovery of phosphoramidite dimers as a means of reducing the costs of oligonucleotide synthesis.