Improving the development of DNA- and RNA-based therapeutics using Bruker’s OligoQuest™ software

Increased use of deoxyribonucleic acid (DNA) and RNA as research agents, as well as pharmaceutical drug molecules has led to oligonucleotide characterization by mass spectrometry (MS) gaining significant interest. The growing importance of oligonucleotides in research, diagnostics and gene therapy has subsequently increased regulatory interest in tools to verify their sequence, as well as identify and quantify related impurities.

RNA and oligonucleotide sequence accuracy is a critical attribute impacting molecular safety profiles to avoid off-target effects or toxicity, and compound efficacy such as correct activity and expression. During the approval process of biopharmaceutical products, characterization of production related impurities or degradation products is of key importance. Axolabs therefore, has developed a unique and proprietary assay system for the sensitive detection of oligonucleotides from biological matrices for the analysis and characterization of drug metabolism and pharmacokinetics (DMPK) and absorption, distribution, metabolism and excretion (ADME) properties of oligonucleotide-based therapeutics.

Though liquid chromatography MS (LC-MS) is an established characterization method, intact mass confirmation of impurities and by-products of oligonucleotides is not always sufficient and 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.

Oligonucleotide MS/MS spectra are complex and manual interpretation is time-consuming and requires in-depth analysis skills. Therefore, the analysis of more than a couple samples a day is difficult, expensive and often results in bottlenecks.

The robustness, sensitivity and high dynamic range of Bruker’s maXis II and timsTOF Pro 2, enables Axolabs to measure biopolymers in the 30-50 kDa range with sub-ppm mass accuracy.

Axolabs also uses Bruker’s OligoQuest™ software to offer enhanced RNA and oligonucleotide characterization for its clients. The team uses OligoQuest™ for confirmation of modified RNA sequences and base exchanges in isomeric oligonucleotides.

Axolabs initially tested the beta version of OligoQuest™, offering enhanced RNA and oligonucleotide characterization for antisense research, RNA therapeutics, confirmation of guide RNA sequences for CRISPR, and other gene editing techniques. Isotopic fidelity combined with ultra-high-resolution MS has shown tremendous promise for these types of analyses. 

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 MS/MS analysis. It also bridges the MS/MS data gap by adding powerful tools for the interpretation of RNA and oligonucleotide data to Bruker’s BioPharma Compass^® suite.

The team at Axolabs sees the relationship with Bruker as a key element to their success, allowing them to chart new territory in DNA- and RNA-based therapeutics and nucleic acid medicines. This includes expanding the use of OligoQuest™ in the company’s other operations, such as its quality control department.

“We’re testing out the timsTOF Pro 2 now, and we have other plans to work with Bruker as we expand our service offerings.”