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Mass Spectrometry In Biopharmaceutical Discovery
- Nov 09, 2018 -

From “www.technologynetworks.com”

Article: May 29, 2018 | by Rachel Brazil, Freelance Science Writer, UK

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.