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Modified Exactive MS Could Enable Thermo Fisher's Entry into Biotherapeutics Characterization Space

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Researchers at Utrecht University and Thermo Fisher Scientific have developed a modified version of Thermo Fisher's Exactive mass spectrometer capable of analyzing protein assemblies as large as one megadalton with single-ion sensitivity.

The work, which was detailed in a paper published this week in Nature Methods, demonstrates that Orbitrap-based instruments could prove useful for analysis of high-molecular-weight targets, a realm of analysis that is currently dominated by QTOF machines, said Albert Heck, chair of the Biomolecular Mass Spectrometry and Proteomics group at Utrecht University and author on the study.

This, Heck told ProteoMonitor, could help Thermo Fisher enter the growing market for biopharmaceutical research and characterization.

In fact, he noted, the researchers currently have another paper under review that is specifically focused on characterizing glycosylation patterns of antibodies – a key part of developing and producing antibody-based drugs like Roche/Genentech's Herceptin or Abbot's Humira.

This second paper shows that "we can really nicely map glycosylation patterns on intact antibodies, and in that way we can really do batch-to-batch control and quality control," Heck said. "I think that for everyone who works on antibodies and needs to do an analytical comparison of their batches or compare their products to those of their competitors, this might be a way to go forward in the future."

Heck suggested that with this in mind Thermo Fisher might look to offer the modified version of the instrument as a discrete product aimed at "the analysis of intact proteins and glycoproteins and other heavily post-translationally modified proteins."

In an email to ProteoMonitor, Andreas Huhmer, Thermo Fisher's marketing director, life science mass spectrometry, said he thought "native MS on an Orbitrap mass spectrometer has the potential to become an essential tool for biopharmaceutical analysis."

He listed as potential applications "characterization of intact biopharmaceutical drugs, including next generation human monoclonal antibodies, therapeutic recombinant fusion proteins, and bifunctional and multifunctional antibodies" as well as ”semi-quantitative analysis of isoforms, undesired impurities such as dimers in drug formulations, and stoichiometric analysis of antibody drug conjugates and other engineered protein constructs, such as pegylated proteins."

The company, Huhmer said, "is actively investigating these applications and others through research collaborations with a number of laboratories and considering future commercialization options.”

Heck noted that he had tried to do similar analysis of large macromolecules on FT-ICR mass spectrometers in the past, but that the machines' magnetic fields made it difficult to keep the molecules in their traps.

An Orbitrap, on the other hand, does not require a magnetic field, making it a potential option for detection of such large targets, he said. And so, in collaboration with Alexander Makarov, Thermo Fisher's director of global research for life sciences mass spectrometry and principal inventor of the Orbitrap, the Utrecht researchers began investigating the company's Exactive machine for this purpose.

Key to making the technique work was adjusting the instrument to remove lower m/z ions, Makarov told ProteoMonitor. "We found that you needed to tune the instrument in a different way to remove all ions below m/z of 2,000 or 3,000, and then the [large] complexes would appear very beautifully in the spectrum," he said.

The researchers also changed the flow ions through the machine, Heck said, noting that in the modified workflow the high-mass ions are transferred to the instrument's HCD cell not to dissociate them but to allow for collisional cooling to improve their transmission into the Orbitrap.

The Exactive was a good candidate for the project, Makarov said, "because it has a very simple ion path… so we didn't need to go deep into the instrument" to make the needed modifications.

The required changes are "mainly changes to the software… and modifications to the gasline that provides gas to the HCD cell," he said. "So there are no changes inside the instrument – the changes are on the peripheral part of the instrument."

Using the modified Exactive, the researchers analyzed a range of targets including native IgG antibodies, bacteriophage HK97 capsid pentamers and hexamers, yeast 2OS proteasome, and Escherichia coli GroEL.

Via these analyses, they found that the Exactive offers significant improvements in sensitivity and resolution compared to QTOFs, said Heck, who also uses QTOFs for intact protein research.

"I don't have a direct number, but I think this is at least 100 times more sensitive and the peaks that we get are much sharper, which means that the experimental resolution we get is way better," he said. "[For instance], we can count how many ATP molecules are bound to GroEL compared to how many ADP molecules. In a QTOF that would be just a single blob. On the Orbitrap you see seven peaks there – that there are seven molecules bound to it."

"So that means you can really start to look at large complexes that are either binding drugs or other small molecules," Heck said.

"[The Utrecht researchers] got really great resolution [using the modified Exactive], and so we can suppose [now] that we can investigate even larger complexes," said Alain Beck, a researcher at Center of Immunology Pierre Fabre and an expert on the use of mass spec for biotherapeutic characterization. "And this opens up a couple of possible research avenues in this field."

"[For instance,] the proof of concept was done on an IgG antibody, which is one 150 kD unit," Beck, who was not involved in the study, told ProteoMonitor. "But some people are now using IgM [antibodies], which corresponds to pentamers of IgG. And so you need to be able to analyze larger complexes… And again, with such technology, you can expect to analyze such a large molecule and see different glycosylation sites and PTMs and other modifications."

The lower resolution offered by QTOFs "is OK for the current antibodies," Beck said. "But for a larger molecule it will be more tricky, and so this will be a good way of solving this issue."

Mass spectrometry "is [now] really mandatory for antibody characterization," Beck said. "And so a lot of people are using these techniques often very early in the process for the antibody lead selection and more and more as a quality control method. Another topic is the advent of biosimilars where we are asking are they really similar or not, and mass spec is the key technology to assess similarity or not of these copies."

"I think it is really a huge competition [in this market] between Waters, Bruker, AB Sciex, Thermo, and even Shimadzu and Agilent," Beck said. "All these vendors really know that biologics are complicated to analyze, and so they are putting a lot of efforts to bring better mass spectrometers on the market. I think it is really an increasing market."

Heck agreed, noting that biotherapeutics firms "like Amgen and Genentech" have heavily invested in mass spec to look at intact antibodies" and that "many vendors like Bruker and Agilent and Waters" have targeted their QTOF instruments at this market.

Such work has been almost exclusively done on QTOFs, Heck said, limiting Thermo Fisher's access. The findings of the Nature Methods study could change that, he said, noting that in addition to offering better sensitivity and resolution, the Exactive was much less expensive than a typical QTOF and so will be "easier to buy for many companies."

Current drawbacks mainly revolve around ease of use of the machine for this purpose, Heck said. "Thermo never being in this market, the software to automatically analyze the data that comes out of [the modified Exactive] is still lagging behind. That is more the bottleneck to bring it to market than the hardware at the moment."

Heck also noted that, while the Exactive offers higher sensitivity and resolution, QTOFs are still able to analyze much larger molecules. "We have data that we haven't published yet where on QTOFs we can see particles of 20 [megadaltons]," he said. "That comes at an m/z of 50,000, and that's not possible on the Orbitrap at the moment."

To date, the researchers have been able to analyze particles as large as 1.8 megadaltons on the Exactive, Heck said.

Makarov noted that his team is now working on improvements in fragmentation of large molecules as well as pursuing improvements in sensitivity by adjusting the pressure in various parts of the instrument.

"This will make it a more and more specialized instrument for these purposes" of analyzing large molecules, he said.

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