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.