Main Conference - Day 3 (May 14)Thursday, 14 May 2026

Phosphorothioate diester linkages play a critical role in the efficacy of many RNA therapeutics. However, there are inherent diastereomeric complexities that these linkages possess ultimately presenting challenges for both manufacturing and analytical characterization. This presentation highlights how Codexis’ ECO Synthesis® Manufacturing Platform enables control over diastereomeric composition, facilitating the scalable production of high-quality siRNA.

Recently, the regulatory landscape for synthetic oligonucleotides has evolved dramatically with the publication of draft guidelines from EMA and CDE. In addition, oligonucleotides will be in scope of the proposed revisions to ICH Q6. This presentation will explore the emerging trends in these regulations, including areas of harmonization and divergence.

Areas for application of platform data and approaches will be discussed. Experience interacting with health authorities will be presented in addition to potential future activities and interactions.

This work demonstrates that Arrhenius-based accelerated predictive stability (APS) can reliably predict siRNA shelf life. APS complements conventional stability studies, with predictions closely matching real-time data. This enables conservative, data-driven shelf-life assessments, supports informed long-term stability decisions, and accelerates development timelines to help siRNA therapies reach patients faster.

This presentation outlines a risk-based strategy for microbiological control in synthetic oligonucleotide drug substance manufacturing. Key recommendations include facility design, environmental monitoring, equipment cleaning, and in-process controls. Emphasizing proactive risk assessment and best practices, the framework ensures consistent production of high-quality oligonucleotide therapeutics and compliance with evolving regulatory expectations.

Robust analytical and process strategies are essential to ensure the therapeutic quality of oligonucleotides modified with phosphorothioate (PS) groups, which are required for nuclease resistance and enhanced efficacy. This PS modification introduces analytical complexity by creating a chiral center and also complicates control strategy because the drug is commonly dosed as a mixture of all its P-diastereomers. It is possible that different diastereomers have different pharmacological properties and different biological activities. Therefore, it is essential to maintain consistent diastereomeric distribution across drug substance batches to justify the consistent efficacy of the drug. While a consistent diastereomeric distribution is typically achieved by using the same coupling activator in solid-phase oligonucleotide synthesis (SPOS), it has been suggested that chromatographic purification of siRNA single strands might separate these diastereomers, altering their ratios. Additionally, it is a regulatory requirement to assess and describe the comparability of diastereomeric profile to justify any changes to the synthetic process. In developing liquid chromatography methods to separate and quantify diastereomers, we found that diastereomer separation is highly dependent on sequence and physical properties, particularly the propensity of single strands to form higher-order structures. Bespoke analytical methods were required for each oligonucleotide, so at AstraZeneca we developed an approach to efficiently develop methods capable of separating and quantifying 2 to 16+ P diastereomers in diverse single-strands and duplexes of siRNA. We also showed that significant diastereomer separation can occur during chromatographic (SAX) purification under certain conditions, potentially causing variability in diastereomer ratio. Characterization of the molecules linked oligonucleotide structure to a higher risk of separation during purification, and thus to a risk of altering the diastereomer ratio. Accordingly, we propose factors to consider when developing a robust, scalable oligonucleotide manufacturing process and mitigations to reduce the risk of changing the diastereomer profile—applicable to SPOS and biocatalytic processes—to enable a seamless clinical program with no risk of changing pharmacological properties between batches of siRNA. Our work provides a case study in robust analytical and manufacturing practices, supporting sustainable, scalable production of oligonucleotide therapeutics. We highlight critical analytical criteria and process recommendations that facilitate seamless clinical progression and regulatory acceptance, ensuring that pharmacological properties remain consistent throughout development and commercialization.

Chromatographic separation of product-related impurities in antisense oligonucleotides (ASOs) is inherently challenging due to the structure similarity among closely related species. Coupling mass spectrometry (MS) to chromatography and UV detection has been a successful strategy to overcome some of the challenges; however, it does not address the need to implement orthogonal chromatographic methods to ensure comprehensive characterization of ASO impurities through distinct separation techniques. In this study, we developed a two-dimensional liquid chromatography (2D-LC) method for the analysis and quantitation of major product-related impurities in ASOs. The method integrates a hydrophilic interaction chromatography (HILIC) in the first dimension with a weak anion exchange (WAX) separation in the second, thus providing high orthogonality based on differences in polarity and charge. We employed Design of Experiments (DoE) to guide method development and systematically optimized key chromatographic parameters in both dimensions. The final optimized method was configured in selective comprehensive mode, and the main peak region from the first dimension was fully sampled in multiple cuts and analyzed by fast WAX separation. The method demonstrated excellent linearity, sensitivity, and quantitation limits. This fully UV-based 2D-LC platform offers a practical solution for ASO impurity analysis without the need for MS detection and analysis, suitable for both development and routine quality control. The ability to switch between HILIC-WAX and HILIC-MS modes enhances the method’s versatility in oligonucleotide characterization and complex impurity profiling.