An Effective Analytical Tool in Biopharma is Mass Spectrometry

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An analytical method called mass spectrometry (MS) determines the mass-to-charge ratio of molecular ions or their fragments. In the mass spectrometer, samples are introduced, ionised, fragmented, and detected in accordance with the molecular mass and signal strength. The structure of large molecules can be examined through fragmentation, as can the precursor molecules and changes, as in the case of proteins. MS is a potent analytical technique for identifying biomolecules and following chemical events and molecular alterations because it allows scientists to pinpoint molecular changes down to the isotopes of individual atoms.

Only the mass-to-charge ratio, or m/z, may be determined by MS alone. As a result, it is frequently employed in conjunction with many different analytical instruments, such as liquid chromatography (LC-MS/MS) or matrix-assisted laser desorption/ionization (MALDI) combined with a time-of-flight detector (MALDI-TOF). From initial target identification and proteomics to toxicity and industrial quality control, MS is utilised for analysis throughout the process of developing biopharmaceuticals, which will be covered in this article.

MS: A crucial method for biopharmaceutical analysis and development

For the characterization of big macromolecules like proteins and DNA as well as for whole-systems research, MS has proved crucial.

Finding the disease’s mechanism, discovering potential molecular targets for treatment, and creating bioactive molecules to act on these targets are the three main components of drug development.

1 MS-based proteomics and chemoproteomics techniques are crucial for the identification and development of new drugs because proteins are the most frequent drug targets and are also being exploited as biotherapeutics. Target deconvolution uses proteome profiling along with affinity probes and other chemoproteomics approaches to pinpoint medication targets and the chemicals that influence their action. The stability of pharmacological targets and their mechanisms of action (MoA) is assessed using thermal profiling in conjunction with high-resolution MS. 2

higher-order structures using MS

Proteomics Large biomolecules, such as biotherapeutics and/or their targets, can be analysed top-down (with little to no fragmentation) and bottom-up (with considerable fragmentation) using MS techniques to discover their main and higher-order structures. This is crucial for figuring out the structure-function relationship between proteins and the mode of action of medicines that are related to them. Coupled MS/MS is particularly helpful in this regard since it can reveal higher-order structure by using sequential fragmentations. These methods are used to evaluate and characterise monoclonal antibodies (mAbs), a class of protein proteins, during their research and commercial production.

Nature’s medicine chest
To create regulated biotherapies, MS can be used to identify the active ingredients in conventional herbal medicines. As a method of quality control, coumarins and other active ingredients in traditional Chinese medicines (TCM) have been quantified using direct analysis in real-time MS (DART-MS). 4 In a different study, the active components of Baihe Dihuang decoction, a specialised TCM remedy for depression and sleeplessness, were examined in mice. Many of the chemicals in the decoction were found to be absorbed by the brain and bind to melatonin and serotonin receptors using LC-MS. 5


Biosynthetic gene clusters that create natural compounds in bacteria and fungi are an alternate source for biotherapeutics. Dr. Neil Kelleher, the Walter and Mary Glass Elizabeth professor of life sciences at Northwestern University, claims that these are nature’s pharmacies. Kelleher’s group runs expression screening on thousands of cultivable microorganisms using ultra-high-resolution LC-MS/MS to find natural compounds and the genes that encode their expression. Over five stereocenters are typical for these novel compounds. They can then be examined using bioassays to determine their outcomes and potential as pharmaceuticals. These techniques in metabologenomics have been employed by Kelleher’s team to investigate the biosynthetic processes for stravidins and biotin. 6


When detecting substances in solutions with other nearly identical molecules, MS is especially helpful. The quantification of mAbs, which are used to treat autoimmune illnesses and are seen in solution with endogenous immunoglobulins, falls within this category. Since high-resolution MS techniques can distinguish between masses down to a few decimal places, they improve solution component identification.

MS-based techniques for clinical research and biopharmaceutical quality assurance

For quality control and toxicological purposes, industrial pharmaceutical manufacture makes use of the high-resolution capabilities of MS. It makes it possible to find undesired byproducts and other impurities during the mass production of biotherapeutics. Additionally, it is used to monitor potentially harmful derivatives and track anticipated drug metabolites during clinical trials. 8


MS is frequently used in proteomics to monitor enzymatic changes to proteins, such as glycosylation and phosphorylation, which can change how a biopharmaceutical chemical works and have negative effects. Similar to proteins, polysaccharides utilised in glycosylation are huge, complex biomolecules that can benefit from size and structure determination by high-resolution MS. MS is used by pharmacology labs and manufacturers to characterise and evaluate crucial quality characteristics for glycosylated biotherapeutics. 9


In a report published in 2022, Dr. Wout Bittremieux used machine learning software to examine MS data from human skin swabs. The Dorrestein lab employs MS to analyse post-translational alterations, produce small molecule therapies, and develop MS methods to structurally identify molecules involved in metabolic exchange. Bittremieux is a postdoctoral researcher in this group. The scientists showed in the paper that some medications taken orally or repeatedly can diffuse through the skin and be found on the epidermis. 10 This demonstrates the potential for using non-invasive MS data from samples for a range of clinical trial uses, such as monitoring the metabolization of medications. “Simple skin swabs can be used to assess treatment adherence,” claims Bittremieux.

Technology advances address MS-related issues.

The time and processing power required for data interpretation and the requirement for sample preparation present difficulties in MS analysis.


The Dorrestein laboratory created the Global Natural Product Social and Molecular Network (GNPS) in 2016 to address the earlier problem.


11 According to Bittremieux, it is an MS data store and analysis platform that is housed on UCSD’s computers and acts as “a search engine for untargeted metabolomics.” Once this data has been reanalyzed and reinterpreted, it can be used by research teams around the world to answer new research questions. The GNPS provided Bittremieux’s skin swab information.

The preparation of samples required for MS systems, which frequently involves purification or separation and results in material loss, is a barrier to the analysis of natural products and subsequent drug discovery. By enabling accurate analysis of small amounts of material, high-resolution mass spectrometry (HRMS) reduces this barrier. It is used to assess metabolites and biomarkers in pharmaceutical whole-systems analysis. 12


Kelleher claims that single-molecule MS, which his team assisted in developing, is the most interesting recent advancement in top-down proteoform detection. The ability to characterise diluted complicated mixtures and do so with the single-molecule resolution has advanced significantly, according to him. Its ability to discern the charge state of each ion, also known as individual ion MS, substantially facilitates the mass assignment of extensively changed proteins, their complexes, and other large compounds.

Future plans for mass spectrometry

Like other analytical technologies, MS equipment is becoming easier to use and more compact, increasing the amount of data that can be collected. Consequently, sample preparation for native-state protein analysis and the development of new natural product drugs becomes simpler.


Thanks to improvements in MS hardware, enormous volumes of data are generated and stored in databases like GNPS. According to Bittremieux, machine learning and deep learning techniques will be used more frequently to process this data. Machine learning models must be able to use enormous volumes of training data, and this is something that the discipline is just now beginning to investigate.

According to him, these models will enable researchers to “truly go deeper into the data” than they could before using more conventional bioinformatics techniques. These methods are already employed in genomics research, and metabolomics and proteomics are swiftly catching up. These data and analyses will produce phenotypic information that will be extensively used in biotherapeutics and precision medicine.

References


1. Meissner F, Bantscheff M, and Geddes-McAlister J. discuss the growing significance of proteomics based on mass spectrometry in drug discovery. doi: 10.1038/s41573-022-00409-3. Nat Rev Drug Discov. 2022;1–18.


2. Tracking cancer medications in living cells by thermal proteome profiling Savitski MM, Reinhard FBM, Franken H, et al. doi: 10.1126/science.1255784. Science. 2014;346(6205):1255784.


3. Srzenti K, Tsybin YO, Fornelli L, et al. Interlaboratory investigation using top-down and middle-down mass spectrometry to characterise monoclonal antibodies 10.1021/jasms.0c00036 J. Am. Soc. Mass Spectrom. 2020;31(9):1783-1802.


4. Zhang Z, Li Y, Yang Y, Tao H, and Liao L: Traditional Chinese Medicine with Coumarins as the Primary Characteristics: Direct Analysis in Real-Time Mass Spectrometry 10.1002/pca.2650 Phytochem. Anal. 2016;28(3):137-143


5. Wu H., Liu R., Wang J., and coworkers The probable active components of the Baihe Dihuang decoction in vivo and in vitro were thoroughly examined using liquid chromatography-mass spectrometry and were verified in silico. 10.1002/jssc.202100434 J. Sep. Sci. 2021;44(21):3933-3958


6. Kelleher NL, Montaser R., “The identification of the biosynthetic process for the biotin antimetabolite stravidins.” 2019;15(5):1134–1140 for ACS Chemical Biology. Reference: 10.1021/acschembio.9b00890


7. Willrich MAV, Barnidge DR, and Ladwig PM Therapeutic monoclonal antibodies are identified and quantified in clinical laboratories using mass spectrometry methods. doi: 10.1128/CVI.00545-16. Clin. Vaccine Immunol. 2017;24(5):e00545-16.


Jiang H, Gao S, He J, Jin H, and Hu G. The application of mass spectrometry imaging technology is an innovation in drug toxicity. 2021;464:153000 Toxicology. Cite as: 10.1016/j.tox.2021.153000


9. Glycoproteomics technologies in glycobiotechnology by Alagesan K, Hoffmann M, Rapp E, and Kolarich D. 2021;175:413–434 Adv. Biochem. Eng. Biotechnol. doi: 10.1007/10 2020 144


10. Bittremieux W, Jarmusch AK, Advani RS, et al. Drug detection in the skin is determined by physicochemical features. 10.1111/cts.13198 Clin. Transl. Sci. 2021;15(3):761-770


Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking Wang M, Carver JJ, Phelan VV, et al. 2016;34(8):828–837 in Nat. Biotechnol. cite: 10.1038/nbt.3597


Advances in high-resolution mass spectrometry applied to medicines in 2020: A brand-new era of information, Géhin C, Holman SW. 2021;2(3-4):142-156 in Anal. Sci. Adv. Reference: 10.1002/ansa.202000149

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