Oligonucleotides are polymeric sequences of nucleotides (RNA, DNA, and their analogs) that are utilized extensively as PCR and microarray-based reagents in life science research and DNA-based diagnostic test kits (target specific primers and probes). With increased frequency, they are being developed as therapeutic drugs for a wide range of disease conditions. While there are just two FDA-approved oligonucleotide-based therapeutic drugs on the market today, there are over 100 currently in the clinical pipeline and many more in earlier stages of development.
Oligonucleotides developed as therapeutics can take a variety of forms – from antisense olignucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), and micro RNAs (miRNAs) that can effect “gene silencing,” which is down-regulating or turning off the expression of specific genes/proteins; to Aptamers that behave like small molecule drugs and bind to specific disease targets; to messenger RNAs (mRNAs) that can be very long, and are being designed to up-regulate expression of a particular protein. To enhance their stability and/or cellular uptake in-vivo, oligonucleotide therapeutics often incorporate chemically-modified nucleotides, are PEGylated, or are otherwise conjugated to other chemical moieties. And like other biologics, the biophysical characteristics and purity of these molecules must be precisely understood and controlled to meet regulatory requirements.
Oligonucleotides are produced through an automated solid-phase synthesis process. Typical lengths range from 20 to 80 nucleotides (mRNAs being an exception, as they can be 1,500 or more nucleotides long). Depending on the application, synthesis scales can vary from nanograms to kilograms. While the synthesis process is efficient, it invariably results in truncated sequences and other process-related by-products/impurities that need to be separated and removed in order to meet purity requirements.
By combining our ACQUITY UPLC instrument technology with our industry leading Oligo Separation Technology (OST) columns, and our high quality MassPrep OST reference standard, Waters brings an unrivaled level of chromatographic performance to oligonucleotide analysis.
To this front-end separation, we offer three fit-for-purpose mass detection and analysis options on the back-end to meet the needs of customers:
Our ACQUITY QDa Detector workflow is our most deployable and scalable solution. It is ideally suited for customers looking to screen a variety of synthetic oligonucleotide samples (including pooled samples) for oligonucleotide ID confirmation and purity assessments.
Our Xevo G2-XS QTof workflow delivers robust high-resolution mass spectrometry (HRMS) performance with isotopic resolution, and greatly enhanced sensitivity for in-depth characterization work, including MS/MS sequence confirmation.
Our SYNAPT G2-Si High Definition Mass Spectrometry (HDMS) workflow is for researchers working at the limits of conventional mass spectrometry who need to further characterize and define their oligonucleotides and associated impurities. Combining high-efficiency ion mobility-based measurements and separations with quadrupole time-of-flight (Tof) mass spectrometry, the SYNAPT G2-Si System enables the analysis of oligonucleotides differentiated by size and shape, as well as mass, to deliver increased selectivity and sensitivity beyond that achievable by conventional mass spectrometry.
All workflows utilize our MassLynx MS software for instrument control and data acquisition, and through our collaboration with Novatia, Ltd., we also offer versions of ProMass and ProMass-HR that have been developed to work seamlessly with MassLynx to enable fully automated data analysis and reporting, including spectral deconvolution of both nominal mass data (QDa) and high resolution data (QTof), target mass screening/oligo ID, and impurity analysis.
To learn more about these workflows and the performance benefits that they provide, please have a look at the following presentation: