Critical Role of Oligonucleotides in Drug Development Highlighted at EAS Session

Oligonucleotides are gaining traction as powerful therapeutic agents in biopharma, showing promising potential in treating genetic disorders, cancers, and viral infections. With this rapid advancement, analytical scientists are focused on developing highly sensitive methods to detect and accurately quantify these complex molecules.

An afternoon session at the Eastern Analytical Symposium (EAS) on November 18 explored the growing role of oligonucleotides in pharmaceutical analysis. Sponsored by the Chinese American Chromatography Association (CACA), the session was led by Yi He, professor at John Jay College of Criminal Justice in New York.

David Zuluaga, a senior scientist at Resolian, opened the session with insights into the challenges and innovative solutions for developing ultra-sensitive hybridization liquid chromatography–mass spectrometry (LC–MS) assays for oligonucleotides. LC–MS assays, he explained, often face obstacles such as nonspecific binding, carryover, matrix effects, and issues with selectivity and sensitivity. Conventional sample preparation methods like solid-phase extraction (SPE) and liquid-liquid extraction (LLE) frequently struggle with low analyte concentrations and high contaminant levels in complex biological matrices, resulting in poor analyte-contaminant separation and compromised robustness and reproducibility.

David Zuluaga, a senior scientist at Resolian, speaking during the session at the Eastern Analytical Symposium on Monday.

Zuluaga highlighted the advantages of hybridization approaches, which utilize sequence-specific probes that selectively bind to target oligonucleotides, enhancing both specificity and sensitivity in these assays.

Following Zuluaga, Xiangji Liu of Frontage Laboratories, presented on bioanalysis of oligonucleotides and their metabolites using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Liu shared three case studies in which his team optimized bioanalytical methods for oligonucleotide analysis, demonstrating practical advancements in this field. He also highlighted the range of therapeutic platforms utilizing oligonucleotides and noted several FDA-approved drugs that incorporate these molecules, including eteplirsen, nusinersen, and patisiran.

Bingchuan Wei, senior principal scientist at Genentech, discussed strategies for probing the structure of single guide RNA (sgRNA) using advanced analytical techniques. Wei noted the challenges of analyzing sgRNA due to its large size, complex impurity profile, and intricate higher-order structures. To overcome these obstacles, his team developed a suite of LC-based methods, including ion-pairing reversed-phase LC (IP-RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEC), and size exclusion chromatography. Additionally, the team employed microfluidic modulation spectroscopy (MMS), an orthogonal spectroscopic technique, to monitor secondary structure changes in sgRNA across temperature variations; sgRNA is a synthetic RNA molecule used in the clustered regularly interspaced short palindromic repeats–associated protein 9 (CRISPR-Cas9) genome-editing system.

The final presentation was given by Vidya Annavarapu from the College of Pharmacy at the University of Georgia, who discussed recent advances in LC–MS analysis of oligonucleotides. Annavarapu and her team compared Waters' OligoWorks and SPE kits with Phenomenex's Clarity cartridges to assess their performance in extracting and quantifying oligonucleotides from rat plasma. The OligoWorks kit includes a detergent-free protocol designed to simplify sample preparation by eliminating the need for evaporation and reconstitution, thereby reducing method development time, she said. The team also conducted a three-day validation study on plasma quality control (QC) standards and evaluated the use of Phenomenex Clarity lysis buffer pretreatment with Waters SPE microplates. Additionally, they compared Phenomenex Clarity SPE cartridges with Waters OligoWorks protocols, focusing on factors such as proteinase K digestion, pKa differences, and method scalability. The Waters OligoWorks solution was further evaluated for oligonucleotide stability and digestion time courses.

As oligonucleotides continue to gain prominence as therapeutic agents for a range of serious diseases, the demand for precise and reliable analytical methods grows. The presentations emphasized that, while challenges remain, advancements in analytical methods—like hybridization LC–MS, microfluidic modulation spectroscopy, and optimized extraction protocols—are paving the way for more efficient drug development and greater therapeutic impact.