Oligo LC-Based Characterization

Oligos, Oligos, Oligos!

Oligonucleotide therapeutics and reagents are everywhere. They are helping the world address previously untreatable diseases, and they are also being used in new ways to sensitively quantify and sequence important translational research samples. Whether it be a CRISPR guide RNA, lipid conjugated anti-sense oligonucleotide, or a specially modified assay probe, these molecules must be tested to ensure they have the right sequence, pinpoint-specific modifications, conjugate moieties and purities.  And when dosed as a therapeutic, they need to be carefully quantified to confirm pharmacokinetic models.  

Join the Waters Chromatography and LC-MS Team and their collaborators from MRIGlobal, Regeneron, and Acuitas Therapeutics in this special episodic, 3 week webinar series to learn more about the science of oligos and the power of LC-based approaches to analyze them. 

EPISODE 1

Part 1: Ultrafast IP-RP using ultra-short columns and new HILIC insights with GTxResolve Premier BEH Amide Columns

In the first part of the presentation, we provide a comprehensive guide to the analysis of oligonucleotides (ONs) using ion-pair reversed-phase liquid chromatography (IP-RPLC) on ultra-short columns (2.1 x 20 mm). We investigate the effect of ion-pairing (IP) agents on ON retention and show that while IP agents significantly affect absolute retention, their effect on selectivity is often minimal. Our results underscore the utility of systematic method development, particularly to investigate gradient steepness and temperature - which have the greatest impact on selectivity - so that resolution can be optimized for both sequence and length variants. We discuss the importance of using low-adsorption column hardware to minimize non-specific interactions, which will improve method robustness and peak shapes. The webinar will provide practical guidelines for the development of robust, reproducible and highly efficient IP-RPLC methods for ONs, which will be useful for industry analysts working on new therapeutics as well as new sequencing and diagnostic reagents.

The second part of the presentation will focus on some new insights into hydrophilic interaction chromatography (HILIC). Indeed, HILIC seems to be a very promising alternative to IP-RPLC. A new column (GTxResolve Premier BEH Amide) will be presented, which allows the development of fast and robust methods. Method considerations (selection of mobile phase additives, gradient optimization) will be discussed in detail. Solutions to avoid the inherent HILIC breakthrough effects (related to solvent/eluent mismatch) will be provided.

Finally, generic (platform) conditions that can be set for the separation of different oligonucleotides will be discussed.

Presenter: Szabolcs Fekete PhD (Consulting Scientist, Cell and Gene Therapy Consumables, Waters Corporation)

Szabolcs worked in the pharmaceutical industry for 10 years before joining the University of Geneva Department of Pharmaceutical Sciences. In 2021, he joined Waters Corporation and now works as a Consulting Scientist. His current interests include separations of new chemical modalities, fundamentals of chromatography, column technology, and method development approaches. His current interests include separations of new chemical modalities, fundamentals of chromatography, column technology, new method development approaches and modeling. He was the winner of the LCGC Emerging Leader in Chromatography Award in 2020 and the HTC Innovation Award in 2022.

Part 2: Characterizing CRISPR-Cas9 Systems: Analyzing CRISPR Components using Ion Exchange and High Throughput IP-RP LC-MS

Learn about leveraging the differing charges of apo-Cas, unbound sgRNA, and ribonucleoprotein (RNP) complexes in order to separate CRISPR components by ion exchange (IEX) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. We will discuss scaling traditional UPLC methods to achieve even higher throughput IP-RP LC-MS analyses.  A workflow was developed to transfer a UPLC purity method for sgRNA from a 100 mm IP-RP column to a 20 mm high throughput column, which has reduced total method times from 30 to 5 min.

Presenter: Rachel Ginther (Senior Manager Biologics & Cell and Gene Therapy, MRI Global)

Rachel obtained her PhD in Analytical Chemistry from the University of Kansas and has worked in the pharmaceutical industry for seven years. She manages programs in biologics and cell and gene therapy analysis and manufacturing and is actively leading the expansion within the organization into biologics.