E24: Chromatographic Methods of Analysis of Oligonucleotides, siRNA, sgRNA and mRNA

One-Day Course
Date to be announce; 8:30am – 5:00pm
Dr. Martin Gilar, Waters Corp., Milford, MA

We describe development of liquid chromatography (LC) methods for analysis of nucleic acid therapeutic compounds. Ion-pair reversed-phase LC (IP RP LC) or hydrophilic interaction chromatography (HILIC) are method compatible with mass spectrometry (MS) detection. Size exclusion chromatography (SEC) and ion-exchange chromatography (IEC) are useful with simple UV detection.  We will describe the method development for analysis of antisense oligonucleotides (ASO), silencing RNA (siRNA), short guide RNA (sgRNA) and messenger RNA (mRNA). In addition, we will illustrate the impact of non-specific interactions with LC metal hardware on oligonucleotide quantitation, tailing, and carryover.

The short course is for analytical chemists who are developing methods for analysis of oligonucleotide therapeutic compounds and mRNA. The course is amenable for both LC beginners and experienced chromatographers who are new to the field of nucleic acid analysis.

  1. Introduction to LC Methods for Analysis of Therapeutic Nucleic Acids
    – IP RP LC – MS compatibility and selection of ion-pairing system
    – HILIC – alternative to IP RP LC (MS)
    – SEC, IEC – new applications

2. Method Development, Optimization, Selectivity
– Selected applications to ASO, siRNA, and mRNA
– Guidelines to mobile phase preparation, impact on retention variability, etc.
– Recommended columns, column longevity, non-specific adsorption

3. Selected Examples of Nucleic Acid Analysis
– Phosphorothioate oligonucleotides
– mRNA analysis, mRNA mapping, poly(A) tail analysis Software for LC MS data analysis
– Software for LC MS data analysis

Dr. Martin Gilar received his Ph.D. in analytical chemistry from Institute of Chemical Technology in Prague (1996). He spent postdoc years in Hybridon Inc. (1996-1998) and Northeastern University in Boston (1998) developing separation methods for antisense oligonucleotides and fraction collector for DNA molecules. Dr. Gilar is a Scientific Fellow; since 1998 he works at Waters Corporation R&D group in Milford, Massachusetts. He has over 30 years of experience in the separation sciences, including chromatography, electrophoresis, and mass spectrometry. He published 70+ peer reviewed papers. He is a recipient of Chromatographic Society Jubilee medal in 2022 and Jaroslav Janak 2022 medal.