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

 

Solutions for oligonucleotide therapeutic analysis

HPLC Columns
Vanquish HPLC Systems
Mass Spectrometry
Software & Data Analysis

Software & Data Analysis

Optimal solutions for oligonucleotide therapeutic analysis

Optimal solutions for therapeutic oligonucleotide analysis
Oligonucleotide AnalysisThermo Scientific InstrumentThermo Scientific HPLC Column
1. Structural CharacterizationOrbitrap Exploris 240 MS or
Orbitrap Eclipse Tribrid MS
DNAPac RP HPLC Column
2. Impurity Identification & Quantitation Using LC-HRAMOrbitrap Exploris 120 MS
Orbitrap Q Exactive Plus MS
DNAPac RP HPLC Column
3. LC–MS Impurity Identification & Quality Control ScreeningISQ EM Single Quadrupole MS
LTQ XL ion trap MS
DNAPac RP HPLC Column
4. High Performance Ion-Pair Reversed-phase LC-UV CharacterizationVanquish Horizon or
Flex UHPLC System
DNAPac RP HPLC Column
5. High Resolution Anion Exchange LC-UV CharacterizationVanquish Horizon or
Flex UHPLC System
DNAPac PA200 HPLC Column
6. Purification & High Load Capacity SeparationsVanquish 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.

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:

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

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.

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.

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).

Aptamer

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
Common chemical modifications used in oligonucleotide drugs

 

Resources

Brochures

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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:

 

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