What motivated you to focus on LC-MS for oligonucleotide analytics?
Melanie: The growing importance of oligonucleotide therapeutics was a key driver. Over the past years, we’ve seen a significant increase in approvals and clinical trials, which also raises the demand for highly sophisticated analytical methods. LC-MS stands out because it allows us to address complex analytical questions—especially when it comes to identity, impurities, and structural characterization.
As a CDMO supporting drug products from early development to commercialization, it was essential for us to invest in a technology that can cover this entire lifecycle.
Why is LC-MS particularly suitable for oligonucleotide analysis?
Melanie: LC-MS combines two powerful techniques: chromatographic separation and mass-based detection. This is particularly important for oligonucleotides because they are structurally complex molecules.
The chromatography step helps separate closely related species, while the mass spectrometer provides detailed molecular information. This enables us to identify, characterize, and quantify oligonucleotides—even in complex mixtures—making LC-MS one of the most versatile tools for this class of molecules.
Which analytical parameters can LC-MS help to assess?
Melanie: LC-MS can cover a broad range of critical quality attributes. These include identity confirmation, purity and impurity profiling, and quantification of the active ingredient. In addition, it plays a key role in structure elucidation, such as sequence confirmation or detection of chemical modifications. Depending on the method setup, it can also support the analysis of degradation products and physicochemical properties.
What are the main limitations of LC-MS when working with oligonucleotides?
Melanie: There are several challenges. First, oligonucleotides often show lower ionization efficiency, which affects sensitivity. Second, they tend to form multiple charge states and adducts, which complicates the spectra.
In addition, chromatographic separation can be challenging and often requires ion-pairing reagents, which may contaminate the system. Data analysis is also more complex due to the structural diversity of these molecules.
Overall, while LC-MS is powerful, it requires careful method development and experienced analysts to generate reliable results.
How does LC-MS support early-stage development of oligonucleotides?
Melanie: In early development, LC-MS is a critical tool for understanding the molecule. It allows us to detect and quantify impurities, identify degradation products, and confirm the correct structure and sequence.
It also supports process development by monitoring synthesis and helping optimize purification steps. This level of insight is essential to build a strong analytical foundation before moving into later development stages.
How do you use LC-MS for structure confirmation or sequencing?
Melanie: For structure confirmation, we rely on high-resolution MS—typically qTOF systems. These allow us to determine the exact mass of the molecule and confirm its molecular composition.
For sequencing, LC-MS/MS is used. The molecule is fragmented in a controlled way, and the resulting fragments are analyzed to reconstruct the sequence. This approach is particularly useful for verifying that the oligonucleotide has been synthesized correctly and contains the intended modifications.
What are the biggest analytical challenges during method development?
Melanie: One of the biggest challenges is achieving sufficient sensitivity and reproducibility. Oligonucleotides are difficult to ionize, and their polarity makes chromatographic retention challenging.
Another key challenge is optimizing conditions to minimize adduct formation and ensure clean spectra. Additionally, method robustness is crucial, especially if the method will later be transferred into a QC environment.
Finally, data analysis can be very complex, so selecting appropriate software tools and workflows is also an important part of development.
What advice would you give to companies starting with oligonucleotide analytics?
Melanie: My main advice would be to take a holistic approach from the beginning. Don’t look at LC-MS as a standalone technique—consider how it integrates into your overall analytical strategy and lifecycle concept.
It’s also important to invest in the right technology and expertise early on. Oligonucleotide analysis is complex, so experienced scientists and well-designed methods are key.
And finally, consider scalability and regulatory requirements from the start. Methods developed in early stages should be transferable to QC environments later on, which will save time and resources in the long run.
