ISC 2026 Update: Nucleic Acids and Oligonucleotides

An afternoon session on Wednesday, September 9, 2026, at ISC 2026 in Prague, Czech Republic, focusing on the analysis of nucleic acids and oligonucleotides was moderated by Davy Guillarme from the University of Geneva, Switzerland, and Koen Sandra from the RIC Group, Belgium.^1,2

Sebastiaan Eeltink from Vrije Universiteit Brussel, Belgium, kicked off the session with a talk on Separation Matters: Advanced Chromatographic Approaches for Impurity Profiling of Emerging Oligonucleotides. This research focused on characterizing therapeutic oligonucleotides (ONs), with an emphasis on analytical challenges associated with phosphorothioate (PS) modifications. These modifications generate closely related diastereomers that cannot be distinguished by mass spectrometry alone, making effective chromatographic separation essential, according to Eeltink.

He described the development of complementary liquid chromatography (LC) strategies for both single- and double-stranded ONs. Anion-exchange chromatography (AEX) was investigated and optimized to improve the separation of closely related species. AEX was subsequently combined with ion-pair reversed-phase LC in a two-dimensional workflow, providing enhanced impurity profiling and improved characterization of therapeutic ONs.

Additional approaches were presented for examining phosphodiester and shortmer impurities, including reversed-phase liquid chromatography–mass spectrometry (RPLC–MS) and chemical derivatization to determine the number of PS modifications.

The presentation also addressed lipid–oligonucleotide conjugates, where the nature and position of the lipid can markedly affect chromatographic behavior. Comparison of reversed-phase and hydrophilic interaction liquid chromatography (HILIC) methods demonstrated the influence of lipid structure on retention and diastereomer resolution. Notably, a C4 stationary phase enabled resolution of more than 30 of the 64 theoretically possible siRNA diastereomers, highlighting the potential of tailored chromatographic methods for complex ON therapeutics, according to Eeltink.

Hailin Wang from the Research Center for Eco-Environmental Sciences, China, presented on Capillary Electrophoresis for Separation and Characterization of Protein-DNA Assemblies and Condensates. This project involved the use of capillary electrophoresis (CE) to study complexes formed between proteins and deoxyribonucleic acid (DNA). These are molecules that vary widely in composition, charge, and size and are therefore analytically challenging to separate and characterize.

Because CE separates species by differences in electrophoretic mobility, it can distinguish free components from assembled species, and Wang’s team applied it to investigate protein–DNA interactions and higher-order assemblies, including condensate-like structures. Wang showed that electrophoretic methods can reveal how protein–DNA assemblies form and differ from one another, while also demonstrating how separation methods originally developed for small charged molecules can be adapted to far more complex nucleic-acid-containing systems.

Martina Lioi from Novartis Pharma AG, Switzerland, spoke on Expanding the Analytical Toolbox: Denaturing Ion-Pair HILIC and RP Approaches for Impurity Characterization of Fatty Acid-Conjugated siRNAs. Lioi highlighted chromatographic approaches for characterizing impurities in fatty-acid-conjugated small interfering RNAs (siRNAs), where the added hydrophobic lipid component introduces structural and chromatographic complexity beyond that of conventional oligonucleotides.

Her group evaluated a denaturing ion-pair form of HILIC, known as IP-HILIC, alongside reversed-phase approaches to obtain complementary separation mechanisms, using both to examine closely related impurities and product variants associated with the conjugated siRNA. The work makes the case for using more than one chromatographic mode when analyzing modified nucleic-acid therapeutics: ion-pair reversed-phase chromatography remains the standard for denaturing oligonucleotide analysis, while HILIC and related approaches can add different selectivity and reveal additional product-related species, according to Lioi.

The session concluded with a talk from Sylwia Studzińska from Nicolaus Copernicus University in Toruń, Poland, on the Development of Sample Preparation Methods for the Extraction of Therapeutic Oligonucleotides from Plasma and Cerebrospinal Fluid. Extraction is a critical step in oligonucleotide bioanalysis because these compounds can bind strongly to proteins and other matrix components.

Studzińska presented strategies for improving sample preparation for therapeutic oligonucleotide analysis. The work focused on antisense oligonucleotides (ASOs), whose strong interactions with proteins can complicate their recovery from biological matrices.

Studzińska compared several extraction approaches, including liquid–liquid extraction (LLE), solid-phase extraction (SPE), microextraction by packed sorbent (MEPS), and dispersive solid-phase extraction (dSPE), for samples such as serum, plasma, and cerebrospinal fluid. Practical limitations of existing workflows include lengthy processing, variable recovery, and the use of non-volatile salts, according to Studzińska.

The presentation also explored new adsorbent materials developed by her research group for hydrophilic-interaction and mixed-mode extraction. These approaches showed potential to simplify sample preparation while reducing processing time and improving extraction performance.

References

1. ISC 2026 Program: https://www.czech-in.org/cmPortalv15/Searchable/isc26/config/normal#!sessiondetails/0000375210_0(accessed 2026-09-09).
2. Holčapek, M.; Nováková, L. ISC 2026 Preview. LCGC International 2026, 3 (5), 34.