Mass Spectrometry in Biopharmaceutical Discovery
Mass Spectrometry in Biopharmaceutical Discovery
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Biotherapeutics now makes up 25% of new drugs approvals. Their efficacy and safety are highly dependent on their structures, which are complex, heterogeneous and subject to modification. Analysis of biological molecules therefore needs a different approach to small molecule pharmaceuticals. Mass spectrometry (MS) has become the go-to technique to supply answers to a range of analytical questions, offering sensitivity, selectivity and specificity. Over the past decade advances in mass spectrometry are allowing more information on higher order structure and new imaging techniques are even providing insights into how biological drugs behave in vivo.
MS can detect, identify and quantify molecules separated by their mass to charge ratio. But biological molecules pose some unique problems. “They are often hundreds of times the size of conventional small molecule drugs,” says Todd Stawicki, biopharma applications scientist at SCIEX. This tends to affect the sensitivity you can achieve with mass spectrometry. Plus, “biosynthesis is highly heterogeneous,” adds Stawicki, “it often creates lots of small variations. Then the analytical challenge is to characterize those small variations and determine if they are clinically relevant.”
Typically, a mass spectrometer ionizes the sample using electrospray ionization (ESI) where a high voltage is applied to a liquid to create an aerosol. This 'soft ionization' technique creates little initial fragmentation – often an issue with large molecules. Ions are separated by acceleration in an electric or magnetic field and then detected by an electron multiplier: the deflection experienced is a function of the mass-to-charge ratio. The mass-to-charge ratio of the ions is determined by one or several different types of mass analyzers. To gain more structural information tandem mass spectrometry (ESI-MS/MS) is used, where discrete ion can be isolated, fragmented and their mass-to-charge ratio determined.
Mass spectrometry and protein structure
The information that can be obtained from a protein is immense, including its primary amino acid sequence, post-translational modifications and even higher-order structure. The most basic information – its amino acid sequence – is routinely found by trypsin digestion to create peptide fragments that produce a fingerprint mass spectrum. Structural information on the various polypeptides within a protein can also be found using methods such as breaking disulphide links using a reducing agent. “With an antibody you can simply reduce them, so rather than one single protein you are going to get four smaller subunits. It can allow you a finer resolution and detail while still maintaining a lot of its intact structure,” says Kelli Jonakin, Senior Global Marketing Manager for Pharma and BioPharma at SCIEX.
“There are also a wide range of different post-translational modifications that can be directly observed by mass spectrometry,” says Jonakin. These are the enzymatic modifications that occur to proteins following biosynthesis and can have an impact on the efficacy and safety of a drug. An important one is glycosylation – the covalent addition of sugar moieties to specific amino acids. Approximately half of all proteins expressed in a cell undergo this modification. Glycans can be enzymatically separated from the protein before analysis to produce a glycosylation fingerprint.1 By engineering a protein’s surface glycosylation pattern, drug developers hope to enhance therapeutic performance.
Mass spectrometry also has a crucial role in detecting contaminants from the bioengineering process says Jonakin: “‘When the engineered cells are producing these biotherapeutics they also produce lots of other proteins (host cell proteins, HCPs) and some of these can have undesirable properties.” Mass spectroscopy is able to characterize and quantify these impurities. “In development, if there is a host-cell protein that is particularly immunogenic and represents a high safety risk, scientists could develop an LC-MS assay to quantitatively monitor a unique signature peptide for that host-cell protein, and then monitor for this in the purification and release batch for the biotherapeutics,” explains Jonakin.
Mass spectrometry and drug efficacy
In biopharma mass spectrometry play a role in measuring drug efficacy often by analyzing downstream effects. “We can employ it for looking at the biological consequence of the drug-target binding,” says Stawicki, “for example, we can use mass spectrometry to look at phosphorylation of a messenger protein caused by the binding event.” But, it is also now possible to probe a biotherapeutic’s interactions with its target by performing a native mode analysis.
According to chemist, Igor Kaltashov, from the University of Massachusetts Amherst, the technique is not yet widely established, but he says: “industry is becoming more interested in this aspect of mass spectrometry. I would say in 2–3 years it will become commonplace.” Native mass spectrometry requires protein assemblies to be extracted from solution into the gas phase using ESI. “You have to do it gently, so you don’t break up these complexes.” To assist the move from solution into the gas phase Kaltashov pioneered a technique that uses size exclusion chromatography.2 This first allows the molecular complexes in solution to be separated from smaller molecules whilst preserving biological activity. “You can inject your complex as it’s formed in a phosphate buffer using a solvent system that isn’t compatible with mass spectrometry and use size exclusion chromatography as an interface,” explains Kaltashov.
Another technique to study conformation and binding is hydrogen-deuterium exchange (HDX) MS.3 If heavy water is introduced in solution, hydrogen atoms will exchange with deuterium. The rate of exchange is characteristic of the degree to which the hydrogen is protected and that provides conformational information. “The binding epitope (i.e. the interface residues), will be shielded from your labeling agent and so once the labeling is completed you can interrogate them and by using standard approaches, determine which residues have not been labeled. They are assigned as the residues that are involved in the formation of those binding epitopes,” explains Kaltashov.
Many types of studies require highly sensitive instruments and Jonakin says the gold standard for protein quantification is the triple quadrupole mass spectrometer (triple quad, TQMS, QqQ). This is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series. Quadrupole refers to it being constructed from four parallel cylindrical rods which control the voltage and allow only ions of a given mass-to-charge ratio to reach the detector. In between the two mass analyzer quadrupoles is a third quadrupole which acts as a cell for collision-induced dissociation. “Triple quads are extraordinarily sensitive and there are many places within the discovery and development pipeline where researchers need extreme sensitivity,” says Jonakin, ‘this can be critical for pre-clinical and clinical studies. But, they do not provide deep MS information, that is what quadrupole time-of-flight MS gives you.’’
Quadrupole time-of-flight MS (Q-Tof MS) is another important high-resolution mass spectrometry method. In a time-of-flight mass analyzer, ions are accelerated in an electric field. The pulsed ions travel in a high vacuum, field-free region and then impact an electron multiplier. Ions travel as a function of their size – small ions travel faster than large ions. “This allows us to detect and characterize proteins and peptides with extremely high accuracy and resolution. That is critically important for confidently distinguishing very small, yet important, differences in very big biotherapeutics,” says Stawicki. These state-of-the-art mass spectrometers can offer a level of detailed information that, for example, enables accurate glycosylation profiling.
For studying biological drugs, Jonakin says Q-Tof MS systems provide a speed advantage: “one of the greater needs for biopharmaceuticals has been speed, pretty much at all levels, because speed equates to depth of coverage and because biologics are so complex you just have so much more going on. The ability to do a lot of experiments in a short time frame allows you to get very deep and very rich mass spectrometry information.”
New data acquisition strategies are also allowing much more comprehensive fragment ion analysis in biological samples using high-resolution QTOF mass spectrometry. This can give you detailed information on every detectable analyte in the sample in a single run. For highly complex biotherapeutics this is a great advantage.
Mass spectrometry and imaging
Another innovation that has proved useful in biopharmaceutical development is imaging mass spectroscopy. One of the earliest methods used for biological imaging was MALDI (Matrix-assisted laser desorption ionization) imaging, first developed in the 1990s by Richard Caprioli at Vanderbilt University. Twenty years later its use is becoming widespread for imaging tissue as part of pharmacokinetic studies. “The thing about MALDI compared to some other imaging MS methods is that it is able to see large biomolecules such as peptide and proteins,” says Adam McMahon, Senior Lecturer in Analytical Chemistry at the University of Manchester.
The method works by covering a thin tissue section with a crystallized matrix material and progressively scanning the surface with a laser. This evaporates the matrix which carries the underlying biomolecules with it. “You generally see intact molecular ions and not their fragments,” says McMahon. “Image files can contain tens of gigabytes of data in which you have peaks at very many masses, each of which will give you an image. So, if you have a drug molecule, you can potentially map its distribution across the tissue sample and can see whether your drug has penetrated into a particular tissue region, which might be important in a tumor, for example.”
Such imaging has been used to explain some unusual off-target drug side-effects, for example MALDI was used to identify the drug metabolite that caused seizures in patients taking an HIV non-nucleoside reverse transcriptase inhibitor.4 “Drug delivery into the brain can also be examined, where the blood brain barrier can exclude certain molecules,” adds McMahon. Others have been using the technique to determine tumor margins, comparing MALDI MS images to histology data.
Another imaging method, desorption electrospray ionization (DESI), instead of using a laser, uses a stream of fast moving charged solvent droplets in the order of 1–5 microns in size, aimed at the sample surface. The droplets extract molecules from the surfaces into the gas phase for analysis. McMahon says there had been skepticism about the spatial resolution the technique could provide, but it can now be as low as 40–50 microns; "You do not see the large proteins extracted in these droplets, you see lipids mainly. But lipids can tell you an awful lot about what is going on in your tissue. You may be comparing a tissue that has and has not been treated with a drug and what you see is the tissue response in terms of the change in the lipid profile."
Mass spectrometry is a crucial tool in the discovery and development of biological drugs but to many is often still seen as a technique for specialists, with results needing complicated interpretation. Developments and subsequent advancements in mass spectrometry mean that the technique is now a lot more accessible to researchers that are less experienced in using the technique.
The power of mass spectrometry is now something that all researchers in biotherapeutics can harness themselves.
1. Viktoria Dotz, Rob Haselberg, Archana Shubhakar, Radoslaw P. Kozak, David Falck, Yoann Rombouts, Dietmar Reusch, Govert W. Somsen, Daryl L. Fernandes, Manfred Wuhrer. Mass spectrometry for glycosylation analysis of biopharmaceuticals. Trends in Analytical Chemistry, 73, 2015, 1-9. https://doi.org/10.1016/j.trac.2015.04.024
2. Characterization of Small Protein Aggregates and Oligomers Using Size Exclusion Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry. Khaja Muneeruddin, John J. Thomas, Paul A. Salinas, and Igor A. Kaltashov. Analytical Chemistry 2014 86 (21), 10692-10699. DOI: 10.1021/ac502590h
3. Detection and Characterization of Altered Conformations of Protein Pharmaceuticals Using Complementary Mass Spectrometry-Based Approaches. Cedric E. Bobst, Rinat R. Abzalimov, Damian Houde, Marek Kloczewiak, Rohin Mhatre, Steven A. Berkowitz, and Igor A. Kaltashov. Analytical Chemistry 2008 80 (19), 7473-7481. DOI: 10.1021/ac801214x
4. Central Nervous System Disposition and Metabolism of Fosdevirine (GSK2248761), a Non-Nucleoside Reverse Transcriptase Inhibitor: An LC-MS and Matrix-Assisted Laser Desorption/Ionization Imaging MS Investigation into Central Nervous System Toxicity. Stephen Castellino, M. Reid Groseclose, James Sigafoos, David Wagner, Mark de Serres, Joseph W. Polli, Elizabeth Romach, James Myer, and Brad Hamilton. Chemical Research in Toxicology 2013 26 (2), 241-251. DOI: 10.1021/tx3004196