The analysis of oligonucleotide therapeutics requires robust and accurate analytical characterization to confirm identity, and to determine purity, quality, and strength. Below are Thermo Scientific optimized solutions for characterizing:
- Antisense oligonucleotides (ASOs)
- RNA interference (RNAi) including siRNA and miRNA
- Aptamers
- Common oligonucleotide chemical modifications
Toolkit
All the tools you need to analyze your oligonucleotide therapeutics
System preparation for optimized oligonucleotide analysis
- Access an optimized cleaning protocol which reduces adduct formation and enhances oligonucleotide LC-MS analysis.
- The system preparation document accompanies a tips & tricks video with further simple, yet essential, tips for oligonucleotide analysis.
Streamlining characterization and monitoring of oligonucleotide impurities using an Orbitrap-based LC-HRAM-MS platform
- Confident base-by-base sequence confirmation and localization of modifications for oligonucleotides and their impurities
- Consistent quantification of FLP and impurities with direct method transfer from discovery to monitoring
Solutions for oligonucleotide therapeutic analysis
Vanquish HPLC Systems
Mass Spectrometry
Software & Data Analysis
Optimal solutions for oligonucleotide therapeutic analysis
| Optimal solutions for therapeutic oligonucleotide analysis | |||
|---|---|---|---|
| Oligonucleotide Analysis | Thermo Scientific Instrument | Thermo Scientific HPLC Column | |
| 1. Structural Characterization | Orbitrap Exploris 240 MS or Orbitrap Eclipse Tribrid MS | DNAPac RP HPLC Column | |
| 2. Impurity Identification & Quantitation Using LC-HRAM | Orbitrap Exploris 120 MS Orbitrap Exploris MX MS Orbitrap Q Exactive Plus MS | DNAPac RP HPLC Column | |
| 3. LC–MS Impurity Identification & Quality Control Screening | ISQ EM Single Quadrupole MS LTQ XL ion trap MS | DNAPac RP HPLC Column | |
| 4. High Performance Ion-Pair Reversed-phase LC-UV Characterization | Vanquish Horizon or Flex UHPLC System | DNAPac RP HPLC Column | |
| 5. High Resolution Anion Exchange LC-UV Characterization | Vanquish Horizon or Flex UHPLC System | DNAPac PA200 HPLC Column | |
| 6. Purification & High Load Capacity Separations | Vanquish Horizon or Flex UHPLC System | DNA Swift HPLC Column | |
Quick start: Oligonucleotide analysis applications
Identification and quantitation of oligonucleotides, impurities, and degradation products
The capabilities of high-resolution accurate mass (HRAM) and data-dependent tandem mass spectrometry (ddMS2) enables confident identification, mapping, and relative quantitation of oligonucleotides, impurities, and degradation products in a single experiment.
High resolution LC/MS analysis of therapeutic oligonucleotides
LC-HRAM is indispensable for determining impurity levels. As several modified full-length impurities, the n − 1 deletion and n + 1 addition sequences often cannot be baseline separated chromatographically and it is therefore challenging to quantitate at low levels.
Oligonucleotide mass confirmation
Expanding a UV quality control method with MS provides mass confirmation and impurity identification.
High performance reversed phase chromatography column for LC-UV characterization of oligonucleotides
The DNAPac RP HPLC column is designed for analysis of oligonucleotides and double-stranded (ds) DNA/RNA fragments using LC/UV or LC/MS. The unique column chemistry provides excellent performance under a broad range of pH, temperature, and mobile phase compositions. In addition, the wide pore size of the resin provides excellent separation of large double-stranded nucleic acids up to 10k base pairs.
Ultra-high-resolution anion exchange LC-UV characterization of oligonucleotides
Highest resolution charge-based oligonucleotide separations. These anion-exchange columns provide unsurpassed resolution of full length from n–1, n+1 (and other failure sequences).
Oligonucleotide purification using Thermo Scientific DNASwift HPLC Columns
The Thermo Scientific DNASwift Monolith Column provides exceptionally high resolution and capacity, laboratory scale purification of DNA and RNA after synthesis. This anion-exchange HPLC column typically results in superior purity and yield compared to other ion-exchange columns. DNASwift is recommended for mg-scale separations of crude mixtures that can be further characterized with the DNAPac PA200 and/or the DNAPac RP products.
Identification and quantitation of oligonucleotides, impurities, and degradation products
The capabilities of high-resolution accurate mass (HRAM) and data-dependent tandem mass spectrometry (ddMS2) enables confident identification, mapping, and relative quantitation of oligonucleotides, impurities, and degradation products in a single experiment.
High resolution LC/MS analysis of therapeutic oligonucleotides
LC-HRAM is indispensable for determining impurity levels. As several modified full-length impurities, the n − 1 deletion and n + 1 addition sequences often cannot be baseline separated chromatographically and it is therefore challenging to quantitate at low levels.
Oligonucleotide mass confirmation
Expanding a UV quality control method with MS provides mass confirmation and impurity identification.
High performance reversed phase chromatography column for LC-UV characterization of oligonucleotides
The DNAPac RP HPLC column is designed for analysis of oligonucleotides and double-stranded (ds) DNA/RNA fragments using LC/UV or LC/MS. The unique column chemistry provides excellent performance under a broad range of pH, temperature, and mobile phase compositions. In addition, the wide pore size of the resin provides excellent separation of large double-stranded nucleic acids up to 10k base pairs.
Ultra-high-resolution anion exchange LC-UV characterization of oligonucleotides
Highest resolution charge-based oligonucleotide separations. These anion-exchange columns provide unsurpassed resolution of full length from n–1, n+1 (and other failure sequences).
Oligonucleotide purification using Thermo Scientific DNASwift HPLC Columns
The Thermo Scientific DNASwift Monolith Column provides exceptionally high resolution and capacity, laboratory scale purification of DNA and RNA after synthesis. This anion-exchange HPLC column typically results in superior purity and yield compared to other ion-exchange columns. DNASwift is recommended for mg-scale separations of crude mixtures that can be further characterized with the DNAPac PA200 and/or the DNAPac RP products.
Products for therapeutic oligonucleotide analysis
Robust High-Resolution Orthogonal Separations
The Thermo Scientific DNAPac family of HPLC columns sets the standard for oligonucleotide purity analysis, fast screening, and purification:
DNAPac PA200 Oligonucleotide HPLC Columns
For strong anion exchange, high-resolution analysis
The DNAPac PA200 columns support screening of synthetic oligonucleotides for production yield and failure sequences on a routine basis; unit-base resolution of synthetic oligonucleotides has been demonstrated for up to 60 bases and beyond. These columns provide unsurpassed resolution of full length from n–1, n+1 (and other synthetic failures) not possible with other columns.
Key benefits:
The DNAPac PA200 column provides industry-leading resolution of therapeutic oligonucleotides, with:
- Separation of oligonucleotides 60 nucleotides long with an average separation speed of between 8 and 9 bases per minute in a 10-minute chromatogram
- Separation of siRNA oligonucleotide strands of identical length, sequence, and mass that differ only by the presence of aberrant 2’, 5’ -linkages along the oligonucleotide, including resolution of such ONs where the position of the aberrant linkage occurs in different positions
- Separation of the Rp and Sp diastereoisomers in ONs harbouring one or two phosphorothioate linkages
DNAPac RP HPLC Columns
For high resolution and high throughput LC-UV or LC-MS analysis
Compatible with MS and separation of large and small oligonucleotides and double-stranded (ds) DNA/RNA fragments, the Thermo Scientific DNAPac RP reversed phase HPLC column has high pH and temperature stability, excellent sample resolution, and exceptional column lifetime. In addition, the wide pore size of the resin provides excellent separation of large double-stranded nucleic acids up to 10k base pairs.
Key benefits
The DNAPac RP reversed phase HPLC column delivers high performance reversed phase chromatography for fast, high-resolution oligonucleotide separation and analysis, uniquely providing:
- Separation of mixed-base ONs on a stable polymeric stationary phase
- Adjustable gradient curve, temperature, and flow to improve resolution and/or reduce analysis time
- Excellent MS compatibility
Advanced LC systems for solving tough analytical challenges
Obtain new benchmarks in accuracy, precision and sensitivity with the Thermo Scientific Vanquish UHPLC systems. Providing biocompatibility with a state-of-the-art quaternary or binary high-pressure solvent blending, these ultra-high-performance liquid chromatography systems share all Vanquish values, such as a design focused on uptime, robustness and reliability.
Vanquish Flex UHPLC Systems
Succeed with flexibility
Thermo Scientific Vanquish Flex UHPLC systems excel from method development to routine analyses. With binary and quaternary pump options, Vanquish Flex UHPLC systems offer outstanding performance for LC and LC-MS based oligonucleotide workflows.
Vanquish Horizon UHPLC System
Delivering results without compromise
For your most complex and difficult oligonucleotide samples, or for applications where you are aiming for the shortest cycle times and highest throughput. The Thermo Scientific Vanquish Horizon UHPLC system affords greater confidence for the most demanding LC and LC-MS based oligonucleotide workflows.
Key benefits
- Better separations
Separate more peaks than ever before. The Vanquish system improves specifications on all fronts. It supports higher backpressures, better thermostatting, optimized volumes, better linearity, and more sensitivity. - More results
Regain time during your projects. The system improves analysis speed, increases sample capacity and has improved robustness. - Easier interaction
The Vanquish system has an optimized design, automated features and works with the operational simplicity of Thermo Scientific Chromeleon CDS software.
Identify and quantify oligonucleotides, impurities, and degradation products
Orbitrap Exploris 240 Mass Spectrometer
The Thermo Scientific Orbitrap Exploris 240 MS delivers the performance and versatility to drive discovery and identification with the quantitative precision and accuracy to confidently scale up while achieving impact. Operational simplicity with intelligent data acquisition helps you discover your fast track to confident results across a range of oligonucleotide-based applications. Using proven Orbitrap mass analyzer technology, you can address any challenge and achieve your objectives with excellence.
This high-resolution accurate mass (HRAM) and data-dependent tandem mass spectrometry (ddMS2) of this MS uniquely enable:
- Rapid assessment of oligonucleotide purity and comparison of different purification methods.
- An in-depth understanding of forced degradations of oligonucleotides.
- Insight into the structure of high MW impurities, which cannot be obtained using the MS1 method alone.
Software and Data Analysis
BioPharma Finder 4.0 software
Streamlined workflow for the characterization of oligonucleotide impurities and degradation products
Thermo Scientific BioPharma Finder 4.0 software introduced oligonucleotide analysis workflows to streamline processes from sequence creation and oligonucleotide mapping to relative quantitation of impurities and result reviewing. The software includes an array of tools for comparative analysis to allow easy optimization of ddMS2 and comparisons of data from different studies, including impurity analysis and time course degradation
About Oligonucleotide Therapeutics
Oligonucleotide therapeutics are short, single- or double-stranded DNA or RNA molecules that bind via Watson-Crick base pairing to enhance or repress the expression of target RNA, in order to treat or manage a wide range of diseases. They include, Antisense oligonucleotides (ASOs), RNA interference (RNAi), and aptamers.
Oligonucleotide and RNA therapies expand the range of drug targets beyond traditional protein based biotherapeutics, to include DNA and RNA:
Guide RNA can be used to directly edit gene sequences, RNAs (asRNA, siRNA, miRNA) target mRNA and ncRNA, RNA aptamers block protein targets.
Click image to enlarge
Therapeutic Oligonucleotide Control of Disease
ASOs match the complementary sequence of a specific mRNA. ASO can have two different effects on the mRNA. Some modifications of ASOs trigger the destruction of the mRNA. This will result in the loss of the corresponding protein. Other modifications can mask only certain parts of the mRNA leading to a modified version of the protein.
Click image to enlarge
RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules
Click image to enlarge
Target recognition and binding of aptamers involves three-dimensional, shape-dependent interactions as well as hydrophobic interactions, base-stacking, and intercalation. Aptamers bind to a portion of their target in which they fit.
To date, oligonucleotide therapeutics have focused on gene silencing other strategies are being pursued, these including gene activation and splice modulation strategies which have the potential to expand therapeutics targets beyond what is generally accessible to conventional pharmaceutical modalities.
There are hundreds of oligonucleotide therapies currently in clinical development with several gaining regulatory approval. Despite this, difficulties remain in achieving efficient delivery to target organs and tissues.
The most commonly used strategies employed to improve nucleic acid drug delivery include chemical modification to improve ‘drug-likeness’, covalent conjugation to cell-targeting or cell-penetrating moieties and nanoparticle formulation, endogenous vesicle (exosome) loading, spherical nucleic acids (SNAs), nanotechnology applications (DNA cages).
Common chemical modifications used in oligonucleotide drugs
In addition to ensuring oligonucleotide therapeutic modifications occurred as intended, one must also monitor for impurities that occur during oligonucleotide synthesis. Such as:
- N-1, N-2 from failed sequences
- N+1
- PO backbone reversion
- De-protection (DMT)
- Deamination of base
- Depurination of base
- Distereoisomer of PS backbone
Resources
Brochures
Webinars
Optimizing Analytical Separations of Synthetic RNA
BioPharma Finder 4.0: Advanced Workflows for Oligonucleotide Analysis
Oligonucleotide analysis: Practical implementation approaches for HPLC and LC-MS analyses
Oligonucleotide analysis, new practical advances and tips to a mature technique
Scientific Articles
Development of an ion pairing reversed-phase liquid chromatography-mass spectrometry method for characterization of clustered regularly interspaced short palindromic repeats guide ribonucleic acid
Application Notes
Brochures
Also see
For oligonucleotides utilized as PCR and microarray-based reagents in life science research and DNA-based diagnostic test kits (target specific primers and probes), refer to:
For phosphoramidites and solid supports for oligonucleotide therapeutic synthesis, refer to:
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