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The Power of Mass Spectrometry in Biopharmaceutical Development

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The rise of protein biopharmaceuticals

The oldest protein biopharmaceutical is recombinant insulin, which was first approved for the treatment of diabetes in 1982. Since then, this class of medicine has diversified to include antibodies, enzymes and other protein-based macromolecules. In 2019, biopharmaceuticals accounted for almost
half of all new drugs approved by the US Food and Drug Administration (FDA), demonstrating the rapid growth of this field over the past 40 years.1 This rapid growth can be partly attributed to the versatility of protein biopharmaceuticals, which find applications as therapeutics in almost every category of disease, from enzyme replacement therapies for rare genetic disorders to antibody drugs for cancer.

The versatility of protein structure

Unlike small molecule drugs, proteins are large, dynamic macromolecules that adopt complex multilevel structures. The structure of a protein can undergo vast alterations in response to minor changes in their environment; for example, antibodies undergo significant conformational changes upon binding to their target. This adaptability is a crucial factor that makes proteins’ diverse biological functions in nature possible. However, it also makes their characterization as biopharmaceuticals challenging. In order to successfully characterize proteins, tools and techniques that are equally versatile are required and this is where mass spectrometry (MS) comes in.

MS is traditionally applied to elucidate the protein contents of a sample according to their mass. In recent years, the information garnered from mass spectra has expanded significantly through innovative new techniques.
Professor Christoph Borchers, McGill Centre for Translational Research in Cancer, states that “mass spectrometry alone is less useful, but in combination with protein chemistry it can be used for determining structural changes.”

Protein higher order structure and dynamics

The higher order structure of a protein therapeutic has a direct impact on its potency and safety and is a key parameter to characterize during biopharmaceutical development. It includes the secondary, tertiary and quaternary levels of protein structure, which determine how the protein functions.

According to Professor Derek Wilson, University of York, Toronto, “(X-ray) crystallography and NMR are the gold standards in terms of high-resolution structure, but these approaches are largely static and proteins don’t actually work like that.” He continues: “The conformational dynamics of a protein are very important to understand how the protein accesses higher energy structures”. These techniques are also limited by other requirements, such as a need for the protein to crystallize or for the protein to be small. When discussing the advantages of MS over other tools, Professor Wilson states that: “with mass spectrometry, we can get moderate resolution structural information with dynamics and we can measure protein binding.”

Hydrogen-deuterium exchange

Hydrogen-deuterium exchange (HDX) MS is a structure-dependent labeling technique that can be used to gain insight about protein structure and dynamics. The protein is produced in water (H2O) and then diluted into heavy water (D2O), before being analyzed by MS. When the protein is diluted into heavy water, the accessible hydrogens contained in the protein structure are exchanged with deuterium. As deuterium is heavier than hydrogen, the exchange increases the overall mass of the protein, which can be measured by mass spectrometry. For each hydrogen that is exchanged, the molecular weight of the protein increases by 1 Dalton. Essentially, HDX-MS measures the kinetics of hydrogen-deuterium exchange in the protein.

A key application of HDX in biopharmaceutical development is epitope mapping, which elucidates where exactly an antibody binds to an antigen. Wilson affirms: “The number one application of HDX at the moment is probably epitope mapping.” When certain regions of an antigen protein interact with an antibody, the solvent exposure of this region is decreased and the HDX rate is reduced, which determines whether there is an interaction or not. By combining chemical crosslinking with HDX-MS, the specific site of interaction can also be resolved. Borchers explains, “We can determine epitope sites using HDX and crosslinking.” In this protocol, two proteins are chemically crosslinked, digested and analyzed by mass spectrometry in order to determine exactly where the antibody-to-protein interaction occurs. Borchers continues: “We have very small cross-linkers to get a high resolution”.

HDX-MS can also be used to prove the likeness of biosimilars. A biosimilar is to biopharmaceuticals what generics are to small molecule drugs. A biosimilar is
defined as a biological product that is highly similar to, and has no clinically meaningful differences from, an existing approved reference product.2 When seeking approval for a biosimilar, a manufacturer must produce comprehensive analytical data that establishes comparability and similarity to the reference product. “It is very important for biopharmaceutical companies to show that the structure is similar. HDX-MS data helps a lot to get FDA approval,” says Borchers. Using HDX-MS the mass shift of the biosimilar and reference product can be investigated, Borchers explains that “the mass shift might be 29% in the original and 30% in the biosimilar.”

Post-translational modifications

Post-translational modifications, such as glycosylation, are a
critical quality attribute (CQA) of biopharmaceuticals.3 Regulatory agencies require data regarding the glycosylation state of protein biopharmaceuticals to ensure the safety and potency of the formulation. However, glycosylation states are not straightforward; one sample of a biotherapeutic often contains a distribution of glycosylation states.

Wilson explains that “with small molecule therapeutics, the molecule you have is the molecule you have, whereas with biologics that is not the case. Samples with specific distributions of glycosylation states may have different effects”. For example, one distribution of glycosylation states could be more efficacious than another. Borchers continues: “Using HDX-MS, we can see what the distribution of glycosylation states is and which distributions are more effective, and adapt manufacturing processes to favor these.”

Identification of contaminants

Most protein biopharmaceuticals are manufactured using recombinant production technology. As these recombinant proteins are
derived from biological sources, such as genetically modified cells and organisms, a key challenge in is the removal of residual host cell or process contaminants, which may persist in the sample if the purification steps are not optimized.4 The presence of contaminants has direct negative consequences for drug quality and efficacy. Furthermore, regulatory agencies specifically require monitoring and control of contaminants and process-derived impurities.

MS can be used to identify contaminants that remain in the sample at each stage of the manufacturing process. The manufacturing process can be
altered accordingly to remove specific contaminants and any impurities. “The real power of mass spectrometry lies in its specificity; if you have a solution with 10,000 things in it, mass spectrometry can technically identify all 10,000 components of the mixture and quantify them, especially when combined with HPLC,” says Wilson.

Intrinsically-disordered proteins

Beyond the traditional paradigm of protein function being determined by its structure, intrinsically-disordered proteins (IDPs) are found. IDPs are highly dynamic proteins that do not require a unique structure in order to perform a function. IDPs are
common in complex organisms,5 and the most well-known may be amyloid-disordered proteins, which are often associated with neurodegenerative diseases. While discussing his work on IDPs, Wilson explains: “You can’t do crystallography or NMR, but you can do HDX-MS. We can characterize these proteins on a millisecond timescale and see what their conformational biases are – the conformational bias explains how the protein responds to different drugs.”

Influencing drug design with protein dynamics

Protein biopharmaceuticals are complex and dynamic structures. The protein structure and dynamics have fundamental impacts on the therapeutic efficacy and safety of the biologic. Static images of a protein’s structure can be obtained using techniques such as X-ray crystallography and NMR, which form the basis of structure-guided design for small molecule drugs. However, when the active ingredient is a protein, which can be differentially modified during production by glycosylation, undergo dramatic conformational changes upon binding its target or even not have a fixed structure at all, dynamics hold the key to understanding the effects of the drug.

In the words of Professor Wilson, “[Thanks to MS] you can adopt a whole new paradigm for drug design – we’re talking about dynamics-guided drug design, rather than structure-guided drug design.” Therein lies the power of MS in biopharmaceutical development.


  1. Novel Drug Approvals for 2019. (2020). Retrieved 26 June 2020, from https://www.fda.gov/drugs/new-drugs-fda-cders-new-molecular-entities-and-new-therapeutic-biological-products/novel-drug-approvals-2019
  2. About Biosimilars and Interchangeable Products. (2020). Retrieved 26 June 2020, from https://www.fda.gov/drugs/biosimilars/biosimilar-and-interchangeable-products#biosimilar
  3. Szekrenyes A, et al. (2020). Quantitative Comparison of the N-Glycosylation of Therapeutic Glycoproteins Using the Glycosimilarity Index. A tutorial. TrAC Trends in Analytical Chemistry. DOI: https://doi.org/10.1016/j.trac.2019.115728
  4. Jozala A F, et al. (2016). Biopharmaceuticals From Microorganisms: From Production to Purification. Brazilian Journal of Microbiology. DOI: https://doi.org/10.1016/j.bjm.2016.10.007
  5. Uversky V N. (2019). Intrinsically Disordered Proteins and Their “Mysterious” (Meta)Physics. Front. Phys. DOI: 10.3389/fphy.2019.00010