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Microflow LC-MS for oligonucleotides analysis

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Oligonucleotides present their own unique challenges for characterization and quantification. They are uniquely susceptible to fairly high levels of synthetic impurities. Using the typical quantification method of ion-pairing reagents can be complicated. A simple way to overcome these challenges is to use a lower flow that maintains system uptime and increases sensitivity.

I recently gave a webinar called “Understanding Oligonucleotides from Every Angle: Utilizing Mass Spectrometry for Characterization and Quantification”. You can watch it on demand, now >

In this blog I will share the Q&A for the submitted questions that we did not have a chance to answer during the live webinar.

Questions and answers to help improve your oligonucleotides analysis:

1. If microflow is good for sensitivity and ion pairing reagent reduction, wouldn’t nanoflow be even better?

If you follow the flow rate versus ionization efficiency curve, then yes. Unfortunately, there are practical limitations. As anyone who has worked with nanoflow will tell you, it can be very finicky and have very limited uptime. Most people working in a quantitative environment can’t tolerate low uptime. In addition, nanoflow is more susceptible to fouling of the fluidic system due to high salt loads. Addressing that robustness and uptime time was a principle goal of the M5 MicroLC and OptiFlow® Turbo V Ion Source. It offers the enhanced ionization efficiency of micro flow rates while still delivering high uptime that is suitable for a quantitative environment. Finally, the chromatographic efficiency of nano versus microflow and high flow will likely result in poor separation of components.

2. You discussed the QTRAP® system for doing method development of a quantitative assay, can you use the QTRAP system for more qualitative work?

Yes. The linear ion trap function of the QTRAP series triple quadrupoles enables fast MS or MS/MS scanning to enable qualitative analysis. While it might not offer the same resolution as an HRMS instrument, quite a bit of qualitative work can still be accomplished on it.

3. Are there any challenges with doing oligo quant with a triple quadrupole?

Of course. There are trade-offs with any MS platform. The challenge for doing MRM-based quantification work on a triple quadrupole is that you need to define your MRM masses.& While this does take a little bit more method development work than a high resolution workflow, you do get the benefit of that extra sensitivity that only triple quadrupoles can provide. This challenge can be minimized by adopting the QTRAP technology.

4. You presented quantitative work on a triple quadrupole and characterization on the accurate mass. Can you do quant with accurate mass?

Yes, you can definitely do quantitative work on our HRMS instruments such as the TripleTOF® 6600 LC-MS/MS System. We will have an application note that highlights that work.

5. Are you going to talk about Morpholinos analysis?

Unfortunately, we didn’t get a chance to talk about that during the webinar. It has been difficult to get access to oligos based on a Morpholino backbone. There is every indication that the same workflows can be achieved on Morpholino oligos, but we have not yet tested them. If you are interested in collaborating, we would happily demo our systems with Morpholinos.

6. What is the resolution for the data shown in the presentation? Thank you!

The MS resolution depends on the specific data to which you are referring. The HRMS data shown has a reported resolution above 40,000. The nominal mass data with the QTRAP system is around 0.2 Da, while the setting used for quantification is around 0.6 Da.

7. Do you have any data showing longer oligonucleotides such as 100mers?

We do not have any data to show here, but we are working with another Danaher company, IDT, to acquire data on longer oligos.

8. What’s your take on using oligonucleotides for cell and gene therapy compared to other more traditional methods?

That’s a big question! In all seriousness, we don’t have visibility into all of the therapeutic strategies being pursued by our customers. I think it is largely an issue of therapeutic suitability. There are certainly a large number of diseases that are best addressed, with the lowest instances of side effects, with cell and gene therapies.

9. Is SPE always better than LLE? SPE is not as robust as LLE based on my experience.

The short answer is “no”. SPE is not always better than LLE. There are definitely times when LLE outperforms SPE. Nonetheless, there are advantages to SPE that make it much more attractive than LLE when SPE is available. It is very easy to use and to execute for larger numbers of samples. You can also achieve concentration (as in the opposite of dilution) of your analytes. In addition, there are many times when it is much more selective than LLE.

10. From the slide of comparison of IS levels, you showed a 10x factor between classic flow and microflow. Did you experience a sensitivity increase?

Yes, microflow flow rates with the OptiFlow Source afford better ionization efficiency and thus better sensitivity.

11. What are your preferred ion pairing agents for oligos?

We typically use diisopropylethyl amine with hexafluroisopropanol. Other suitable amines also perform quite well, such as triethyamine and hexylamine, among others.

12. Do you have experience doing purity analysis of longer oligonucleotides – 100mers — more catered towards CRISPR Cas9 applications?

We do not have any data to show here, but we are working with another Danaher company, IDT, to acquire data on longer oligos.

13. Did you also quantify plasmid?

Plasmids are generally too large to yield spectra that are “clean” enough upon which to do quantification. However, CE-LIF on the PA 800 Plus Pharmaceutical Analysis System is ideally suited to quantification of these larger nucleotide structures.

14. Is there a generic IS oligo that can be applied to the quantification of diverse oligos?

My take is that there is no “silver bullet” for oligonucleotide internal standards. There is always going to be a trade-off between the accuracy of an internal standard and its convenience. For instance, a stable isotope labeled internal standard is always the best, but they are inconvenient and only perfectly applicable for a single oligonucleotide. I suggest the key thing is to examine the multitude of oligo analytes you expect to quantify and choose the most common structural features and then base your internal standard on those. Different structural features are going to impact the ionization and fragmentation of oligo analytes. For instance, is the backbone all phosphonothioate? How many of the residues are locked? Are there a lot of 2′ O-methyls? Once you have determined that, go with the structural features that are most representative. You might not get away with a single oligo standard, but you might be surprised by how few you actually need.

15. Can shorter lengths be captured in the SPE procedure, like 3-mer, 4-mer?

Yes. Shorter oligomers can also be captured, but it will depend on specific conditions and oligo chemistries.

16. Does ProMass have any 21 CFR Part 11 compliant features (e-signatures, audit trail, data read/write controls, etc.) for use in a GMP environment?

We are not aware of any compliance features in ProMass.

17. Do you think microfluidic LC-MS will replace the traditional LC-MS for oligonucleotide quantification? Other than better sensitivity, will this be a really good cost saving method?

There are clear benefits to using microflow LC-MS for oligo analysis including better sensitivity and reagent cost savings. The big drawback to using microflow has always been the lack of robustness and lack of stationary phase availability. Fortunately, we believe we have largely solved both of those issues with our M5 and OptiFlow Source solutions. Nonetheless, there are always going to be people who are comfortable with high flow and want to stick with it, and that’s fine. That’s why we offer solutions that work for either flow regime. In fact, that is one of the key features of the OptiFlow Source. You can run it high flow and if you decide you want to make the jump to microflow, you just have to connect a different LC. No big deal.

18. Could you go over some of the best practices in terms of MS cleaning, if an instrument cannot be dedicated for oligonucleotide work?

The big challenge is really with the LC rather than the MS. MS fouling is largely attributed to poor sample prep and inadequate removal of matrix components. We find that for those with robust sample prep, the MS cleaning is only needed every 6-12 months, depending on use (besides the curtain plate which is cleaned every 2-4 weeks). With respect to LC cleaning, this is a challenge. The nature of the ion pairing systems make them quite pervasive in the LC even with the best cleaning. They will adhere to pump seals, o-rings, etc. In addition, even the best LC platforms have some unswept volume which makes complete removal very challenging without a PM and significant flushing.

19. For the 0.1 ng/mL LLOQ, what matrix is used? Is it plasma or tissue?

The matrix in all of the examples shown was plasma.

We hope our answers have been helpful to you! If you have any further questions, please post them in the comments.

About the author
Sean McCarthy, Ph.D.
Global Technical Marketing Manager, Biopharmaceuticals
Sean M. McCarthy received his degree in Chemistry from the University of Vermont and has held several scientific and business development positions to address pharmaceutical and biopharmaceutical characterization. Sean is currently the Business Development Manager of Biologics at SCIEX, where his focus is on delivering targeted solutions for biopharmaceutical characterization, with emphasis on process analytics and development.


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