Taking Advantage of Measuring Molecular Collisional Cross-Sections in Pharma​

insights from industryMike Greig Director of US Pharma/BiopharmaBruker Daltonics

In this interview, News-Medical Life Sciences talks to Mike Greig, Director of Pharma at Bruker, about why measuring collisional cross-sections is beneficial to developing new drugs.

Please can you introduce yourself and tell us a bit about your role at Bruker?

My name is Mike Greig. I have been at Bruker for almost three years as Director of Pharma/Biopharma for the Americas. My job is to align the different parts of the life sciences mass spec division to make sure that we are serving pharma the best that we can.

Can you explain what the collisional cross-section of a molecule is and the theory behind it?

The collisional cross-section of a molecule is a measurement of a molecule in the gaseous phase. If you take the surface area of the molecule and rotate it around into a sphere, the volume that it would occupy ends up being the collisional cross-section. It is a physical property of a molecule in the gas phase. Once you can measure it, you have become CCS-aware..

Across science and industry, what applications can benefit from exploiting collisional cross-sections?

As far as applications go, for anything you would think to measure on a mass spectrometer to get a molecular weight, there is no reason why you would not want to measure additional physical properties such as the collisional cross-section.

Collisional Cross-Section Measurements in Pharmaceuticals

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Why is collisional cross-section such an important concept in Pharma?

For pharma, in particular, it spans the world of molecules from the small-molecule pharma to large-molecule biopharma. Looking at biologics, our collaborators have acquired new data showing that we can obtain exciting information about antibody-drug conjugates by exploiting collisional cross-sections. For small molecules, we can separate chemicals with very small differences in molecular structures, including isomers.

CCS values are interesting, but how important are they for compound characterization in drug discovery? How do these play into the other kinds of data and what does it add to the study?

The CCS value, the collisional cross-section, is an intrinsic property of the molecule, and the more ways that you have to describe a molecule, the better. Having another physical property to describe a molecule enables you to have much higher confidence in identification of a molecule.

One of the things that is really important in pharma is to have confidence in the data that you present to your project teams. Using the timsTOF Pro, you get the molecular weight, the isotopic pattern, the MS/MS data, and now you have a collisional cross-section of each molecule. It is just another way to identify your molecules with confidence.

It is even more important for people doing an API (active pharmaceutical ingredient ) analysis to be really sure that what is in the area under the curve is only a single compound. These are labs that are making kilogram batches of compounds for clinical trials, and they have to be sure they completely understand purity.

By having a collisional cross-section value from ion mobility analysis, you can have more confidence that not only have you chromatographically isolated the product, but you also do not have a mixture of diastereomers or isomers.

Image Credit: klyaksun / antishock

How does collisional cross-section differ between the different pharmaceutical substances you work with?

In general, to measure the collisional cross-section you need a large enough difference in the molecular structure to cause that collisional cross-section to be different. Our instruments - the timsTOF Pro, timsTOF Flex and the timsTOF - all have high-resolution ion mobility capabilities.

We have seen differences that you would not expect to be separated such as stereoisomers. Other examples include cross-linked molecules which have a signature pattern, protein oligomers, as well as anti-body drug conjugates.

Image Credit: Jaroslav/ Shutterstock.com

Can you talk to us about generating collision cross-section libraries for pharmaceuticals and their uses?

There are a lot of different ways you can go about using CCS values. There are many groups compiling libraries of CCS values and this is being done, not just with the Bruker timsTOF Pro line of instruments, but with other types of ion mobility instruments too.

Because our instruments obtain accurate CCS values, we can utilize CCS libraries obtained from drift tubes, the ion mobility standard. Many of our collaborators are generating extensive CCS libraries, including everything from small molecules to lipids to peptides. These CCS libraries can be cross-referenced just like you would reference a peptide sequence in a proteomics database.

Is it necessary to have empirical CCS libraries in order to take advantage of ion mobility, especially when thinking of basic interpretations and predictions for lipids and peptide scoring?

To start with, yes. One of the nice things about these libraries is that many of them are or will be publicly available. For example, we are working with the Scripps Research Institute in San Diego to generate hundreds of thousands of CCS values for small molecules. There are also a growing number of predictive programs available for a wide variety of molecular types where you won’t need empirical libraries. You can tackle the identification and matching of CCS values in multiple ways.

How do Bruker Daltonics take advantage of collisional cross-section measuring in other areas of life sciences, such as immuno-oncology?

Several groups are studying immunopeptides in relation to immuno-oncology. Our collaborators at Monash University in Australia, are using the timsTOF Pro to identify and catalog more immunopeptides faster than with previous instruments. This is because of the ability to customize methods based on peptide shape and charge.

Image Credit: Shutterstock.com/Idwar

Is there a benefit to using ion mobility in situations when chromatographic separation is not possible such as MALDI or other high throughput methods?

There is a huge advantage. Whether you are using MALDI, where you have no separation, or if you just want to use a very short LC method where you have very limited separation, trapped ion mobility provides greater depth of analysis.

It is almost like doing 2D chromatography, but the second stage is in the gas-phase, so no complex LC set-up.

If you are doing spatialOMx starting with MALDI mass spec tissue imaging experiments on the timsTOF fleX, TIMS is really important because there is no separation on the front end. If you have a complex mixture with MALDI, now you have the ion mobility factor to separate these compounds to give you a much more confident analysis or identification of whatever molecules you are looking for.

When acquiring CCS values are other parameters such a resolution, speed or sensitivity affected?

In mass spectrometry, especially in pharma, it is all about speed. Mixtures are getting more complex, and so you need to be able to have more data points per second. You do not want to have to run a three-hour proteomics method when you need to run 100 samples per day. The speed of the timsTOF Pro without losses to sensitivity or resolution are key.

What are the benefits and limitations to different collisional cross-section measuring methods for determining the CSS for different kinds of substances?

There are many groups using machine learning based on huge datasets to create predictive algorithms. We can predict collisional cross-sections extremely accurately for lipids, peptides and small molecules. As we feed these programs with more data, the accuracy will improve, and more importantly, the variety of molecules such as post-translationally modified peptides will be added.

Trapped ion mobility can be used to obtain highly reproducible collisional cross-section (CCS) values. Can you tell us about the TIMStof instruments and the expertise Bruker has in this area?

Number one priority for pharma is robustness. We have found that the robustness and reproducibility on the timsTOF instruments has been outstanding.

Next is the ability to do these ion mobility experiments without a loss in sensitivity. The timsTOF instruments operate in a way we get almost 100% duty cycle, meaning we detect almost every ion entering the mass spectrometer. This is also done at full speed of the instrument, so no loss in MS/MS speed or even resolution. Since it is based on our TOF technology, we also get excellent ion statistics, meaning that we get an accurate isotopic envelope, which can be used for confirmation of molecular formula.

The incredible speed of the instrument allows us to consider doing things like a CE/MS, capillary electrophoresis MS. This involves peak widths of maybe a second, which is too short for many mass specs to collect useful data, but with the timsTOFs, you can easily collect 100 MS/MS scans across that one second. Prior to this, with slower instruments, this was not possible, and so this may enable whole new ways of thinking about chromatography on the front end.

globalImage Credit: Vit-Mar / Shutterstock.com

Ion mobility and collisional cross-section (CCS) measurements have been around for quite some time. What new and attractive features does the timsTOF Pro bring to the field?

In addition to measuring a robust and reproducible collisional cross-section, it is very sensitive. We do not lose sensitivity when measuring the collisional cross-section, so that makes it very different. In fact, the overall design of the instrument, including PASEF (parallel accumulation, serial fragmentation) allow for both speed and sensitivity. Some labs have identified hundreds of proteins from as little as 4 cells with rapid analysis.

How do you know you are measuring CCS values reproducibly and accurately?

One of the things I do when I am presenting to people is show them a map of the globe. We have timsTOF Pros in our applications lab in Bremen, Germany and are working with the ANPC (Australian National Phenome Centre) in Perth, Australia who also have a timsTOF Pro. That is about as far apart on the globe as you can get. We measured the same molecules at each site, and found the variance was less than 1%.

We have also done this experiment with our other demo labs across the globe - Shanghai, San Jose, California and Billerica, Massachusetts. We still get the exact same reproducibility and robustness of our CCS values.

What is the future of collisional cross-section measuring in Pharma and what part will Bruker Daltonics play in this?

I think that this is really exciting. The timsTOF Pro is one of the reasons that I joined Bruker from pharma three years ago. When I talk to people about this, I say, "With collisional cross-section, it is now a physical property we could measure routinely and robustly. So, what are we going to do with this?" Different people come up with different ideas.

With immunopeptides, you get very minimal data from MS/MS, but now we have collisional cross-section we can use CCS with accurate mass to identify a peptide sequence. That is really something that could not be done before. Each conversation with these different groups in pharma provides different ways exploit CCS.

At the end of some of my talks about collisional cross-section, I show a picture of the Nobel Prize and say, "Hey, what are you going to do next with the collisional cross-section?" I am not guaranteeing a Nobel prize for anybody, but it is something to think about!

About Mike Greig

Mike Greig joined Bruker in 2018 as the Director of the US Pharma/Biopharma Business Unit for Bruker Daltonics. In the previous 20 years, he worked at Pfizer in Drug Discovery - most recently leading the Protein Dynamics Group, a core biological mass spectrometry research group focusing on Oncology. During his two decades at Pfizer, he directed labs performing everything from high throughput analysis of small molecule libraries using supercritical fluid-MS, protein NMR, native mass spectrometry, HDX-MS for structural biology, protein turnover, fragment-based drug design, to proteomics. He also spent several years at Ionis Pharmaceuticals managing an oligonucleotide based mass spectrometry research lab and worked at Revlon Science Institute as a polymer and analytical chemist. He has taught over 25 mass spectrometry classes at various scientific conferences and companies worldwide, was a Keynote speaker at the International Mass Spectrometry Conference, a member of the Lab Automation Scientific Committee (now SLAS), member of National High Magnetic Field Laboratory FTICR MS Advisory Panel. Mike has over 50 scientific publications ranging from PK properties of oligonucleotides in mice, native MS of biological complexes, HDX to identify resistance mechanisms in oncology, to SFC/MS of small molecule libraries.

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