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New Technology Aims To Accelerate Biopharma Development

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Biotherapeutics (or biologics) are drugs that are produced using living organisms. Examples include cell therapies, proteins and antibody-based therapeutics. Developing and manufacturing biotherapeutics at scale requires thorough analysis of the active substance’s characteristics to ensure it fulfils both efficacy and safety requirements. Analytical technologies, such as mass spectrometry (MS), are key tools adopted in this process.

Thermo Fisher Scientific recently announced the launch of a new Direct Mass Technology mode, a feature that equips their ultra-high resolution mass spectrometers with charge detection. The addition of this individual ion technique will enable researchers to analyze biotherapeutics, protein complexes and viral particles that were previously too complex to resolve. 

To learn more about Direct Mass Technology, how it compares to traditional methods and the promise it holds for the development of next-generation drug modalities, we spoke with Andreas Huhmer, Senior Director, Omics Marketing, Life Science Mass Spectrometry, Thermo Fisher Scientific.

Ash Board (AB): How does Direct Mass Technology achieve speed, sensitivity and resolution when analyzing complex protein mixtures?

Andreas Huhmer (AH):
It all has to do with the complexity of the mixture that you're analyzing. What you are doing essentially, is capturing the ions that come in through the mass spectrometer in the front end in your Orbitrap and then, instead of looking at what we call the beat, which is the frequency that those ions rotate, or orbit around the orbitrap, we look at the amplitude. So, we don't do anything different in that sense, we just look at different parts of the signal. That allows us to understand the charge of the individual molecule species and that's what we essentially read out for the Direct Mass Technology mode. On a higher level, you can now imagine, if you have limited space, and the Orbitrap has a limited space, like every other three-dimensional container, the more you put different molecular species in there, the more you limit sensitivity. The time you need to record becomes a little bit longer for a single ion than for 10 ions of the same molecular species. We speed this up by multiplexing the measurement and measuring multiple ions at the same time. So, it all comes down to the complexity of the sample. At this point, Direct Mass Technology is not developed for very complex mixtures, you wouldn't infuse the whole proteome for example. At this point in time, you focus on simpler mixtures, typically complexes, purified antibodies, something along those lines. They are still mixtures, as we now see from the signal, but they're not the typical proteome mixture.

AB: Can you discuss how the Direct Mass Technology can gather so much data from extremely small amounts of sample?

With Direct Mass Technology, we inject fewer ions into the Orbitrap than we would for typical m/z measurements, also referred to as ensemble measurements. As you look at the signal amplitude of the individual molecular species, you record every time they orbit the central electrode, what the amplitude of the single signal of individual molecular species is and you can do this in parallel for all the molecular species that you have captured. Essentially you can add the signal up that then represents the charge for individual molecular species. So, it's really a single ion molecule measurement that you are accomplishing. But you do this in parallel with many ion species that you have in your trap. That is essentially enabled on one of the instruments, the ultra-high resolution mass spectrometer and it allows people to measure both m/z and the charge directly. The difference between doing this with the Orbitrap versus other instrumentation that can perform charge detection mass spectrometry is that we essentially do both measurements at the same time in parallel.

AB: How do the results compare to traditional measurement techniques? 

In traditional measurements, you measure mass divided by charge. The physical principle underlying that measurement is the frequency with which those ions orbit the central electrode. You deconvolute that frequency signal and then you get a mass divided by the charge. That typically gives you these distribution curves of charged species in your sample and, from the difference in the peaks, you calculate what the charge of the molecule is and that provides the mass. In this particular case, where we use Direct Mass Technology, we are directly measuring the mass. That allows you to overcome the resolution limitations you might encounter in traditional measurements, particularly at very high mass for very large molecules. The most high-resolution mass spectrometer on planet Earth cannot resolve, for example, the molecular charge species of a virus or adenoviral capsid. In this case, we can determine the charge directly and can therefore resolve all the molecular species. That's a big breakthrough because up until this point, we had no idea how complex and heterogenous those fractions of viruses or molecular complexes were.

AB: How does the workflow of Direct Mass Technology compare to traditional mass spectrometry techniques?

Traditional mass spectrometer settings for things like shotgun sequencing is a reverse phase liquid chromatography separation column, then eluting the sample. In this case, we can still do LC-MS, but we use mostly in native mass spectrometry conditions, utilizing different types of chromatography separation modes, such as ion exchange and size exclusion. The sophistication of the laboratory typically determines whether they use online or direct infusion of the sample. If they use online, then they can do a typical LCMS set up. The more complex the sample, the longer you need to measure and that means you probably want to infuse the sample. So, do you have sufficient time to gather sufficient signal? As I said, you need to cover all the molecular species in that fraction or in that sample and that takes a longer time, because you need to record all the amplitudes individually, this is reduced in a less complex sample. So that gets back to your question, what's the speed? It's dependent on the heterogeneity of your sample. 

AB: When analyzing ions individually in parallel, how does the data handling compare to traditional measurement techniques?

In traditional measurement techniques, you create a composite signal of all the molecular species. In Direct Mass Technology, you create a signal for each molecular species in parallel, and that creates what we call a STORI plot (selective temporal overview of resonant ions) displaying the detection time vs signal amplitude, whereby the STORI plot slop is proportional to the charge of the molecular species detected. This enables you to look at the individual molecular species you have recorded. Whereas in the traditional mass spectrometry signal, you look at m/z and its intensity.

AB: Key applications of Direct Mass Technology include glycoform analysis, membrane proteins and complexes and biotherapeutic characterization. Can you discuss its utility across these areas?

Each of those categories have their own challenges and you would apply Direct Mass Technology for various reasons. If you're looking at an antibody and you would like to understand the heterogeneity or the purity of your antibody preparation, that may have to do with the glycosylation status of your antibody. If you consider an adeno-associated viruses (AAV), you might want to understand the heterogeneity of the AAV assembly and its cargo. If you want to look inside a capsid to understand which of the viruses contained the load and which do not. In that particular case you would apply the technology to say, this fraction of my preparation contains a load and this one does not carry the load. These are all the ways that Direct Mass Technology will have a big impact. To this day, it has been very difficult to determine whether a particular capsid has the right load or any load at all. Currently, people use ultra-centrifugation and sedimentation to separate and understand if the capsids they want it to load with a particular content contain this or not and if it does have this content, does it the right content or has it been modified? With Direct Mass Technology, because you are directly measuring the mass all of this can be measured directly as the mass is indicative of the container plus the loaders. 

AB: Are there any customer stories that you can share?

As we developed the technology over the last year or so we had around 10 beta customers. As this is the first time a commercial product was available for such measurements very few people have ever seen a single ion measurement using Orbitrap instrument. Even for the most seasoned scientists, they had to look at this twice to appreciate what they are looking at.

When I first looked at some of the samples we had from working together with a pharmaceutical company, they were giving us some of their preparations. We were like, “Wow,” we had no idea how heterogenous the sample was. We showed an example from a commercial preparation of a therapeutic FC-fusion protein, Etanercept (Enbrel®) at a user's meeting. That's a safe drug and is very well characterized. But when you actually use the Direct Mass Technology, you can discern more than 100 different molecular species. That's explainable because Enbrel is known to have at least three N-linked glycans, and at least 13 O-linked glycans. But it's a very different world when you see the composite signal versus the signal of the individual molecular species, because now you can actually quantitate and discern those individual molecular species and that's just mind boggling.

AB: Were you surprised by the data and the levels of insights you were achieving with Direct Mass Technology or is this what you're expected to see? 

AH: I was actually very surprised the first time because I had not expected the heterogeneity of the signal. But I immediately realized the potential for this technology, because as we develop the fourth generation of drug modalities, which are going to be gene therapy, virus vehicles and entire cells. The vaccines that were developed for COVID are a very good example of one of this next generation drugs. These are much bigger, more complex molecules and, in general, are much more difficult to analyze. So, this technology is necessary to truly understand what we are looking at when we make those next generation drugs. Also, when you consider that we are making a lot of progress understanding biology from a molecular perspective in the context of complexes. How do proteins assemble to build in a functional subunit? With the advance of cryo-electron microscopy we know what they look like but we really don't know how they assemble and how the biology happened. For example, with this technology we can now look at how small ligands bind to an ion channel. Ion channels are typically in a membrane and when they're in a membrane, they're very difficult to analyze by traditional means, as they're large, very complex and heterogenous. Now, with Direct Mass Technology, you can actually observe how, for example, a particular drug binds to an ion channel and does its action. This is absolutely game-changing.

Andreas Huhmer was speaking to Dr. Ash Board, Editorial Director at Technology Networks.