ISC 2024 Session Focuses on Cutting-edge Techniques in Oligonucleotide Analysis

A session on the third day of the International Symposium on Chromatography (ISC) 2024 focused on cutting-edge developments in oligonucleotide analysis, an important class of biopharmaceutical therapeutics.

Torgny Fornstedt from Karlstad University, Sweden, delivered a presentation on the "Separation of Next-Generation Therapeutic Oligonucleotides Using Ion-Pair Chromatography Based on Fundamental Separation”. Fornstedt’s team addressed the intricate challenges of purifying therapeutic oligonucleotides, such as allale-specific oligonucleotides (ASOs) and small interfering ribonucleic acid (siRNA), which have grown in prominence due to their potential to treat previously incurable diseases. Fornstedt discussed the complexity of these molecules, particularly their synthesis and the generation of hundreds of impurities, in contrast to traditional small molecule drugs. His presentation focused on optimizing ion-pair reversed-phase liquid chromatography, emphasizing the importance of operational parameters such as the choice of stationary phases, mobile phase composition, surface chemistry, and gradient conditions. He also introduced automated methods for decomposing complex sample peaks to aid in more efficient quality control processes, which could reduce reliance on mass spectrometry (MS) in certain cases.

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Anthony Ehkirch from Novartis Pharma AG, Switzerland then gave a talk entitled “From LC–UV to MS-based Methodology for the Analysis of siRNA.” Ehkirch’s presentation focused on alternative approaches for siRNA analysis, particularly when ion-pair reversed-phase liquid chromatography (IP-RPLC) struggles with separating impurity groups. Ehkirch explored complementary techniques, including anion exchange chromatography (AEX) and hydrophilic interaction liquid chromatography (HILIC), which allow for impurity separation without the need for ion-pair reagents, making them MS-compatible. Ehkirch noted that HILIC could sometimes replicate the separations achieved by IP-RPLC while avoiding contamination from ion pairs, which is critical in siRNA analysis. Ehkirch’s presentation highlighted the importance of modern chromatography techniques in supporting comprehensive analytical control strategies, emphasizing that HILIC and AEX methods could enhance impurity profiling in oligonucleotide therapeutics.

Jufang Wu Ludvigsson from AstraZeneca, Sweden presented on “Separation Method Development of Oligonucleotide on Mixed-Mode Column and by 2D–LC–UV–MS. This study explored the application of mixed-mode chromatography as an alternative to the conventional ion-pair reversed-phase liquid chromatography used for oligonucleotide analysis. This mixed-mode technique, which combines charge-based and hydrophobic separations, demonstrated stronger retention and more efficient separations compared to traditional C18 columns, according to Ludvigsson. Ludvigsson’s emphasized this research demonstrated that this method eliminated the need for ion-pair reagents, although the use of phosphate buffer created MS detection challenges. To address this, the team implemented a two-dimensional LC–UV–MS setup, where the second dimension employed a MS-compatible mobile phase. This approach significantly increased MS signal intensity, allowing for the characterization of the main peaks and impurities, according to Ludvigsson.

Daniel Meston from Gustavus Adolphus College, United States continued with a presentation on the potential of two-dimensional liquid chromatography (2D-LC) for resolving oligonucleotide impurities. In this presentation entitled “Development of 2D with IP-RPLC and HILIC to characterize Impurities in Therapeutic Oligonucleotide Analysis.” Meston described a systematic method development workflow, combining ion-pair reversed-phase and HILIC separations to achieve improved resolution of complex samples. He showcased results where a careful tailoring of elution conditions led to the successful separation of closely related impurities. Meston’s group also demonstrated the value of 2D-LC in accurately quantifying impurities down to 0.3% and identifying them using MS detection. Meston highlighted that optimizing diluent conditions was crucial for achieving high-quality separations and addressed some of the key challenges that arise in 2D-LC workflows for therapeutic oligonucleotides.

Ken Cook from Thermo Fisher Scientific, United Kingdom, provided a thought-provoking presentation called “Dispelling The Myths of Oligonucleotide Chromatography” that dispelled some of the misconceptions surrounding ion-pair reversed-phase chromatography (IP-RPLC) for oligonucleotide analysis. Cook argued that many of the long-standing assumptions about this technique, particularly regarding MS quantitation challenges, are outdated. Cook pointed out that the introduction of sulphur into phosphate groups for stability creates additional issues with stereoisomers, complicating the separation process. Cook also discussed common problems such as amine ion pair and metal adduct, which can lead to multiple split signals in MS analysis. His presentation emphasized the need for a better understanding of the IP-RPLC mechanism to improve the accuracy and reproducibility of oligonucleotide separations. He also shared new insights into sequencing longer oligonucleotides using a simplified method that minimizes contamination, making MS quantitation more straightforward.