BioPharma Compass®

OligoQuest™

Fully automatic confirmation of sequences based on intact mass and MS/MS

Introduction

OligoQuest

Mass Spectrometry for Oligonucleotide Analysis

The growing importance of oligonucleotides in research, diagnostics and gene therapy, including tRNA, siRNA, and other modified oligos up to 120 bases, increased the regulatory interest in tools to verify their sequence as well as identify and quantify related impurities. Sequence accuracy of RNA and oligonucleotides is critical to ensuring the correct activity and expression as well as avoiding off-target effects or toxicity to patients. However, current Next Generation Sequencing methods do not offer comprehensive characterization of highly modified RNA-based therapeutics.

Quadrupole time of flight mass spectrometry (QTOF-MS) instruments are being used to characterize highly modified oligonucleotide sequences as they can determine monoisotopic masses of intact oligonucleotides and their fragment ions with high mass accuracy.

The intact mass confirmation of impurities and by-products of oligonucleotides is not always sufficient, therefore information at the nucleotide sequence level is required. MS/MS experiments coupled with enzymatic digests for longer RNA molecules are needed to fully establish the molecular identity of an LC-MS peak. Because oligonucleotide MS/MS spectra are complex, their manual interpretation is time-consuming and requires in-depth analysis skills, rendering the analysis of more than a couple samples a day difficult, expensive and often resulting in bottlenecks.

Deconvoluted spectrum for a sgRNA and associated low level impurities.

OligoQuest

OligoQuest™ - Part of BioPharma Compass®

Bruker’s OligoQuest™ software offers enhanced RNA and oligonucleotide characterization for antisense research, RNA therapeutics, confirmation of guide RNA sequences for CRISPR, and other gene editing techniques. By exploiting the high isotopic fidelity and mass accuracy of Bruker’s maXis II and timsTOF platforms, OligoQuest™ enables identity confirmation by intact mass and MS2 analysis. Customers are using OligoQuest™ for confirmation of modified RNA sequences and base exchanges in isomeric oligonucleotides.

OligoQuest™ offers algorithms and workflows to annotate tandem MS data from oligonucleotide sequences >100 nucleotides. Dan Fabris, CEO of RiboDynamics and Harold S. Schwenk Sr. Professor at the University of Connecticut, explained: “Isotopic fidelity combined with ultra-high resolution mass spectrometry has shown tremendous promise for the analysis of highly modified RNA, but commercial software tools have been unavailable. Simplifying this analysis with OligoQuest™ will dramatically accelerate research and development in the RNA space.”

The OligoQuest™ workflow in BioPharma Compass® is wizard driven to simplify the method setup and accommodates customer-defined sequence definitions to easily include the wide range of modified nucleotides used by the Pharma industry.

 

A flexible sequence editor supports natural nucleic acid building blocks as well as custom backbone, base and sugar modifications. Proven algorithms for peak detection and deconvolution of MS and MS/MS data obtained with high isotopic fidelity and ultrahigh resolution ensure correct mass assignments even in high dynamic range samples.

Sequence verification of a 75-mer oligonucleotide by LC-MS/MS with OligoQuest™

Oligonucleotide Sequence Verification

OligoQuest

Oligonucleotide Sequence Verification

The timsTOF line of mass specs or the maXis II, paired with the VIP-HESI source for high sensitivity in negative mode, are ideal to analyze heterogeneities in oligonucleotides such as siRNA but also in larger molecules such as single guide RNA (sgRNA) used in CRISPR based gene editing.

Confident mass assignments in complex spectra with True Isotopic Pattern and SNAP enable confident sequence verification even for base substitution resulting in only 1 Da mass differences.

The confident annotation of complex overlapping spectra is also essential for MS/MS based sequence confirmation. OligoQuest™ combines the SNAP deconvolution and advanced sequencing algorithms from RiboDynamics (embedded in BioPharma Compass®) to take full advantage of the maXis II’s and timsTOF’s data quality to annotate terminal and internal fragments.

Oligonucleotide By-Products

OligoQuest

Oligonucleotide By-Products

Oligonucleotide by-products may include scrambled variants and Cytidine-to-Uridine conversions with +1 Da molecular weight differences to the target product sequence, respectively. These can be difficult to characterize solely based on their intact mass.. Information at the nucleotide level is required.

Oligonucleotide MS/MS spectra are complex and their manual interpretation is time-consuming and requires in-depth analysis skills, which renders the full analysis of several samples a day in the routine lab difficult and expensive.

The OligoQuest™ workflow in BioPharma Compass® provides an easy to learn tool for the interpretation of oligonucleotide MS and MS/MS spectra. The high isotopic fidelity of Bruker´s High-Resolution Accurate Mass (HRAM) QTOF and timsTOF

AutoMS/MS spectrum of the 24mer with fragment ion annotations in OligoQuest (A). The Sequence Map (B) displays the 5´- (red) and 3´-fragments (blue) including the observed ppm mass errors. The theoretical fragment ion masses including the matches with the spectrum are also displayed (C). The analysis is summarized in tabular form (D).

instruments, together with the established SNAP algorithm for monoisotopic peak picking, provide reliable annotation of precursor and fragment ion masses. MS and MS/MS spectra are thereby matched with high certainty to sequence candidates such as the target sequence and expected by-products. OligoQuest™ works with user-defined sequence nomenclature thus avoiding a translation of lab-specific sequence definitions to a proprietary software format. Together with the proven workflow wizard and the simple reporting of results in BioPharma Compass®, OligoQuest™ will help accelerate the characterization of DNA or RNA oligonucleotides towards the throughput required by the Pharma industry.

 

 

LC-MS analysis of the 24mer autoMS/MS dataset (A) The oligonucleotide was separated from all impurities, solely salt and ion pairing agent adducts were observed in the 24mer peak. Detected by-products are listed as well (B).

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OligoQuest

More Information

Webinar from April 5, 2022

OligoQuest™: New oligonucleotide characterization workflows with LC-MS (/MS)

Oligonucleotide characterization by mass spectrometry has gained significant interest recently with the increased use of DNA and RNA as research reagents as well as pharmaceutical drug molecules. Characterization of production related impurities or degradation products is of key importance during the approval process of such biopharmaceutical products. Well established tools for this characterization process are LC-MS based methods. During this live from the lab session we will show live how Bruker’s high resolution mass spectrometers is fit for the purpose of analyzing oligonucleotides. We will demonstrate the newly developed software OligoQuest™ which simplifies the characterization of oligonucleotides and oligonucleotide degradation products.

 

Literature on OligoQuest

 

For Research Use Only. Not for use in clinical diagnostic procedures.