Impurities in Synthetic Oligonucleotides: Sources, Analysis and Control
Roumen Radinov, Ph.D.,
Vice President, Process Sciences, Alnylam Pharmaceuticals
Impurity control in oligonucleotide manufacturing is a complex challenge and many factors, including the nature of the starting materials and the specific manufacturing processes used, must be taken into consideration.
Roumen Radinov, Vice President, Process Sciences, at Alnylam Pharmaceuticals, spoke about impurity control at TIDES oligonucleotides and peptides conference in May. He explained that starting materials are a major source of impurities in oligo production.
“Impurities in starting materials are controlled through starting material specifications and by the drug substance manufacturing process,” Radinov said.
The ICH Q11 guideline, which covers drug substance development and manufacturing, is an excellent starting point for any impurity control strategy, according to Radinov.
“According to the general principles for selection of starting materials in ICH guidance, the justification should include a proposal to control impurities.
“This should include both starting arterial specifications as well as control of impurities by the oligonucleotide manufacturing process,” he said.
Radinov added that the control proposal should be supported by detailed experimental data showing that any impurities in the starting material will not impact product quality.
Under the ICH Q11 guidelines, developers also need to provide details about the suppliers and contractors, Radinov said. “CMO management strategies should be included to ensure that pre-GMP manufacturing steps have no impact on drug substance quality.”
Reactive vs. nonreactive
More generally, oligonucleotide developers need to think about potential impurities in the right way before developing any control strategies.
“For starting material impurities, as well as other small molecule impurities, there are two ways of looking at these impurities as reactive and nonreactive,” Radinov said.
“The nonreactive impurities will not react with the oligonucleotide, and they will be purged by the manufacturing process as well as the downstream purification.
“On the other hand, reactive impurities have options to react with process reagents during the synthesis process. They can be purged by the oligonucleotide purification process, but if they’re not, they need to be specified in the starting materials,” he said.
Toolbox
Developers have multiple tools to remove impurities, which include everything from washes during solid phase synthesis that target reagents to later ultrafiltration steps that eliminate chromatography solvents and buffer salts.
The big question lies in how to combine them into a successful control strategy, according to Radinov, who stressed that any such effort should begin with analysis.
“To develop an effective control strategy, impurities need to be identified in the starting material. Then, the individual impurities are independently synthesized, and their structures are confirmed,” he said.
“Those that can be controlled by purification processes are confirmed through spike and per studies. Impurities that cannot be controlled by the purification process are then specified in the starting material using specified limits.”
Developers also need to consider whether a particular starting material is an off the shelf product – where the manufacturer is responsible for controlling impurities – or a bespoke substance, which will require additional work, Radinov said.
He cited GalNAc as an example of a custom manufactured starting material used by Alnylam Pharmaceuticals Inc. and many oligonucleotide companies. GalNAc is manufactured through a proprietary technology and requires extensive characterization and justification.
“Impurities in custom starting materials are typically controlled by vendor and manufacturer specifications but also by the drug substance manufacturing,” said Radinov.
