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In 1998, the US Food and Drug Administration (FDA) approved fomivirsen as the first therapeutic oligonucleotide therapeutic. This approval marked a revolution of mechanism of action discovered decades before finally coming to fruition. Since then, the landscape of chemical modifications of oligonucleotides, conjugations and formulations has evolved tremendously, contributing to improvements in stability, efficacy and safety. Today, more than a dozen antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) drugs are on the market, most of which are designated as orphan drugs for treating rare genetic diseases.¹

Alongside the oligonucleotides, another revolution related to analytical solutions took place. Instruments needed to keep up with the ever-decreasing concentrations of analytes of more potent drugs, pushing the boundaries of sensitivity.

Liquid chromatography (LC) coupled to triple quadrupole technology and accurate mass spectrometry (MS) play an important role during bioanalysis to accurately identify and quantify the oligonucleotide drug and metabolites.

In a recent webinar, available on demand, Shane Karnik (Director of Laboratory Operations) and Troy Voelker (Director of Laboratory Operations) of Aliri Bioanalysis provide valuable insights based on their experiences with oligonucleotide bioanalysis using triple quadrupole and time-of-flight (TOF) instrumentation.

This blog summarizes the answers Shane and Troy provided for the pressing questions asked by attendees during the webinar.

Contact us to learn more.

LC considerations

What type of column do you use for the analysis of oligonucleotides to optimize peak shape, retention and carryover effects?

Troy Voelker: This is a common question, and I hate to give a general answer to it: I do not have one column that I go to every single time. I use the best column for the oligonucleotide that we are analyzing. Multiple vendors have specialty columns for this type of work, and I recommend going through that suite of options. There is a general consideration when looking for a column in terms of chemistry: You want a column that is going to be stable above 65°C, especially when you are analyzing siRNA, since you must elevate the temperature to melt the double strands. The high temperature can cause degradation over time for some column chemistries, which you want to avoid picking. We look at columns from different vendors and decide based on peak shape and carryover. You will have to do those experiments up front to figure out which column is best for your oligonucleotide.

Triethylamine (TEA) is known to be an interference in positive ionization mode. Did you experience any issues when using the same system in positive mode?

Troy Voelker: We really don’t see interferences. That is not necessarily because TEA isn’t interfering. It’s most likely because we have removed it from the system. We only have a couple of systems for which we go back and forth between different ionization modes. This is based on the type of oligonucleotide we are analyzing, such as going from siRNA or ASOs to morpholino oligonucleotide types. When we intend to switch ionization modes, we like to flush the LC systems quite a bit to remove ion impairing reagents. Ion pairing reagents like to aggregate. Therefore, when doing this type of analysis, it is crucial to keep your LC systems fresh and not use old ion pairing reagents. When switching modes, I recommend going through a flushing protocol in between.

Experimental design and method development

What is your recommendation for oligonucleotide quantitation on the ZenoTOF 7600 system? Do you usually use TOF-MS or MS/MS? 

Troy Voelker: Both modes work for quantitation. Which one is better suited for your project is something you want to explore in your method development stage. There are 2 main factors to keep in mind: intensity and signal to noise. Your MS2 data is not going to have the same intensity as your MS1 data. The main reason for that is you are breaking the precursor apart into multiple fragments and therefore spreading the intensity across those fragments. On the other hand, you may achieve a better signal-to-noise ratio for a fragment compared to the precursor. That’s often the case because of filtering out a lot of the background when selecting a particular precursor for fragmentation.

When working with samples that contain metabolites that may start creating interferences by overlapping charge state envelopes, you may want to pick specific fragments that would be unique to the metabolite and the parent, respectively.

The fast acquisition rates of the ZenoTOF 7600 system allow for collection of both, full scan MS1 and multiple full scan MS2, while achieving the desired amount of data points across the peak for accurate quantitation. Since you can get both sets of data, you could still decide later which data set to use.

Could you please explain the terminology MRM^HR in more depth?

Shane Karnik: Let’s first clarify the terminology “multi-reaction monitoring” or, in short, MRM.  When using a triple quadrupole system, you select a precursor m/z in the Q1, fragment it in the Q2 and then select a defined product m/z for the Q3. MRM means monitoring that defined transition on a triple quadrupole instrument. MRM^HR stands for the high-resolution version of an MRM.

Users might be familiar with the terminology “parallel reaction monitoring (PRM),” which is essentially the same thing. The first 2 steps are the same as for a regular MRM experiment: you choose a precursor m/z and fragment the precursor in the collision cell. Then, instead of focusing on 1 fragment m/z, you are doing a full scan of the fragment ions. This means you are going to pick up all the product ions within a defined mass range.

The beauty of this is that you don’t lose any resolution or sensitivity while getting a full set of data. You can mine that data later in different ways: you can isolate the data of a product ion for quantitation or sum multiple product ions to potentially increase sensitivity. The latter is one of the reasons why TOF systems can be quite in line with the sensitivity of triple quadrupole systems. You can also investigate what else is in the data set: for instance, you can mine the data retrospectively to look for metabolites or biomarkers.

The way I think of it is that MRM is a snapshot, while MRM^HR is more of a movie providing more details.

Are you monitoring for depurination when doing MS method development?

Troy Voelker: Yes. Depurination is not anything you expect to see in your samples. It is an artifact usually caused by MS source parameters. It therefore really comes back to what Shane mentioned about the importance of source parameter optimization, such as source temperature and declustering potential. We do this early in the method development and do not look for depurination in the actual samples later.

Did you get the same charge states for your oligonucleotides when using HRMS and triple quadrupole instrumentation?

Troy Voelker: The charge states can be source-dependent to some degree. However, we have found them to be more dependent on the ion pairing agents. When we use the same ion pairing agents on different types of instruments, we tend to see similar distributions across charge states.

We might adjust the ion pairing agent based on the charge state distribution we want to achieve. Trimethylamine, for instance, creates lower charge states, which can fall outside of the range of a triple quadrupole. In that case, we focus on ion pairing reagents that create higher charge states.

What internal standard are you using for LC-MS? Can you share general considerations for selecting internal standards for oligonucleotide quantitation?

Shane Karnik: For the work I presented, a structural analog internal standard with 2 more bases than the analyte oligonucleotide was used. Personally, I have never come across a project for which we got a stable isotope labeled internal standard created. Usually, the internal standards were based on alternating bases, additions or eliminations of bases, resulting in a different m/z than the analyte.

As a general consideration, I recommend picking an internal standard that very much acts the same way as your compound. You want a standard that has the same extraction efficiency and the same ionization. Since you are going to look at peak area ratios between your analyte and standard, changes in responses like enhancements or ion suppression should be consistent between analyte and standard. We therefore do not use a universal standard.

When we work on a client project with several drug lead candidates, we can take another candidate and use that as the internal standard.

Troy Voelker: I agree with Shane: a stable labeled internal standard is rare. The standard is almost always an analog. To add onto Shane’s recommendations, if you are dealing with an oligonucleotide drug candidate with special chemistry, you may want to keep that consistent for your analog selection. In addition, I recommend using internal standards that are slightly higher in mass. With that potential, interferences with metabolites can be avoided. Metabolites such as N-1, N-2 may cause interferences with an internal standard that is lower in mass. We therefore do tend to go for a higher mass internal standard.

When you quantity metabolites, do you typically use reference material to quantify against or do you use the parent?

Shane Karnik: There are 2 answers to this question. When doing regulated GLP work, you must have a well-characterized reference material for that metabolite. However, for early discovery work, when we are just looking for metabolites and want to get a general idea of the concentration of metabolites, we do not need a reference material. We can use the TOF-MS data, look for expected metabolites and extract those m/z from the data. For quantitation, we then assume a similar response as the parent or the internal standard and use the parent quantitation curve for the metabolites. This would be only for non-regulated discovery work.

Troy Voelker: I can add that there are likely going to be differences in the ionization between the parent and the metabolite. It is something that we generally observe. As Shane stated it, the approach without reference material gives you an idea of the concentrations, but you are certainly not going to get an absolute concentration.

A TOF system for oligonucleotide characterization and bioanalysis

You mentioned several instrument features. Could you please elaborate on which features are most impactful for quantifying oligonucleotides and why?

Shane Karnik: First, I want to mention that you really must look at everything comprehensively. That includes the extraction, your mobile phase composition and your mass spectrometer, since it all plays together.

I recommend optimizing the mobile phase additives so that you only have a few charge states for the precursor to avoid spreading the signal over multiple m/z.

Regardless, whether you are using a quadrupole or a high-resolution system, you must optimize your front-end parameters to achieve the best sensitivity. You want to optimize especially your iron spray voltage and your declustering potentials to break up any adducts. Oligonucleotides tend to form metal adducts and those non-covalent interactions need to be broken up for the highest sensitivity.

When it comes to specific features, to me the big differentiator on the ZenoTOF 7600 system is the Zeno trap. When you enable it, you can get up to 95% of the ionizable species into the TOF, whereas with other TOF systems without that trap, you are only getting around 20% duty cycle. Since material is usually limited, it is really important to us to tune the mass spec, use the Zeno trap and only put in the needed amount of material.

Does the ZenoTOF 7600 system allow for characterization of the oligonucleotides by analyzing the fragments? Is there oligonucleotide sequencing software available to process the data?

Shane Karnik: Yes. You can use the MS/MS data from the ZenoTOF 7600 system and process it with Molecule Profiler software to achieve matching of fragments and confirm sequences of synthetic oligonucleotides. If you are trying to identify N-1, N-2, etc. metabolites, you can also use that approach to figure out from which end you are losing nucleotides to form the metabolites. We do that kind of work for reference material. Sometimes we only get UV spectra for these materials, which is not enough detail when we start development. The software helps us with that.

Future outlook: large RNAs

Do you have any experience in quantitation of large oligonucleotides, such as guide RNA (gRNA), and could you provide any recommendations?

Troy Voelker: We have non-MS approaches for the quantitation of gRNA, and we are also investigating quantitation with LC-MS. We think it is feasible and there are publications on this topic. Hopefully, we can provide an update soon. Stay tuned.

¹ Collotta, D. et al. Antisense oligonucleotides: a novel frontier in pharmacological strategy. Front Pharmacol. 2023 Nov 17;14:1304342. doi: 10.3389/fphar.2023.1304342. PMID: 38044945; PMCID: PMC10690781.