Main Conference Day 2Wednesday, 12 November 2025 - CET (Central European Time, GMT+01:00)

Both early and late phase process optimization play critical roles in successful project management. Early phase projects are often driven by speed and therefore the focus of optimization is either on new, often challenging, building blocks or on new chemistries that deviate from standard oligonucleotide synthesis protocols. Conversely, late phase optimization is driven by quality factors, cost reduction, scalability and efficiency. At this stage plenty of process data and real-world evidence are available. Therefore success rate of process enhancement is based on actual performance metrics.

2’-N-methylacetamide (NMA) nucleotides are a novel type of monomer for splice modulating Antisense Oligonucleotides (ASOs). Recently, a sequence consisting of 18 NMA nucleotides was developed and manufactured at scales up to 163 mmol. Despite the seemingly inert nature of the 2’ NMA functionality, it is prone to engaging in side reactions that result in the formation of late-eluting impurities (LEI), which are challenging to control without negatively impacting coupling completion. Ultimately, LEI were mitigated by judicious choice of the amidite to activator ratio used in coupling. Targeted capping steps were introduced in select elongation cycles that were prone to generating deletion impurities, thereby reducing the incidence of deletion impurities with minimal introduction of capping related impurities. Additionally, it was necessary to develop a new cleavage and deprotection protocol to control the rate of NMA related cleavage products, as well as impurities related to the addition of methylamine to the ASO. Described herein is a summary of development efforts and the first on-scale manufacture of a 2’-NMA functionalized ASO.

At Wave Life Sciences, stereopure oligonucleotides containing chimeric phosphorothioate (PS), phosphoryl guanidine (PN) and phosphodiester (PO) backbones are rationally designed to optimize pharmacology and efficacy. Our methods enable synthesis of stereopure oligonucleotides with PS and PN linkages, therefore the configuration of each linkage must be confirmed as part of identity testing. We will describe approaches to confirm stereochemical identity of the oligonucleotide with an emphasis on confirming configuration using chromatography and enzymatic digestion in addition to the standard techniques of Mass ID and MS/MS sequencing.

Oligonucleotide therapeutics, expanding from rare diseases to more prevalent conditions, have increased demand for GMP-manufactured oligonucleotides and acetonitrile. The acetonitrile supply chain is volatile due to economic, regulatory, and environmental factors, affected by crises such as the 2008-2009 financial crash and COVID-19. Addressing these issues necessitates strategic planning, recycling method diversification, and regulatory compliance. A risk assessment process evaluated potential waste contamination in recycled acetonitrile, categorizing contaminants by severity and probability.

Solid-Phase Oligonucleotide Synthesis (SPOS) relies on large wash volumes between reagent deliveries to prevent reagent mixing, in large part due to the poor flow characteristics, and resulting reagent mixing, arising from the intentional presence of a headspace during column packing. Dynamic Axial Compression (DAC) columns are commonly used in chromatography to eliminate headspace in packed columns, making the application of this technology to SPOS a logical opportunity to reduce solvent consumption. This presentation will describe Ionis' successful work to scale DAC column operation up to multi-kilo scale production and reduce total solvent wash consumption by ~25%, with no negative impact on yield or purity.

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The development of siRNA fixed-dose combinations (siRNA FDCs) presents intrinsic formulation and analytical challenges. Key hurdles include distinguishing and quantifying the two siRNAs and their product-related impurities. To assess purity and accurate quantification, orthogonal chromatographic techniques like ion-pairing reversed-phase chromatography (IP-RP-LC) under both denaturing and non-denaturing conditions, as well as denaturing anion-exchange chromatography (AEX), are employed. Mass spectrometry plays a crucial role for the identification and peak purity evaluation. Establishing a comprehensive analytical toolbox for both physical and chemical characterization is essential to assess the feasibility of FDC formulations. By thoroughly designing the analytical assessment strategy, the quality and efficacy of siRNA FDC drug products can be assured.

Method transfer case studies for the oligo assay/impurity methods by IP-RP-UHPLC will be presented. The learnings will include how to address the issues associated with the sensitivity, resolution, linearity, and method precision during the method transfer on different LC systems (Vanquish, Agilent and Waters) at different CDMOs. The cases indicate it is crucial to establish the procedures for the LC system cleaning/maintenance to ensure the successful method transfer and routine use later.