Mass Spectrometry in the Biopharmaceutical Pipeline

Mass Spectrometry in the Biopharmaceutical Pipeline Biopharmaceuticals are booming. In recent years, 40–50% of newly approved drugs have been biopharmaceuticals, including antibodies, polypeptides and drug conjugates.1 However, .1 biopharmaceuticals and biotherapeutics are produced using living systems, adding a level of complexity and heterogeneity not seen in chemical pharmaceuticals. This can pose challenges during all stages of discovery and development, from initial pre-clinical research to manufacturing quality control. Mass spectrometry (MS) is a powerful analytical tool used throughout biopharmaceutical research and development for techniques such as characterization and purity testing. This infographic will explore the role of MS in the biopharmaceutical industry and highlight how MS-based techniques are used throughout the biotherapeutic discovery and development pipeline. What is mass spectrometry? MS is used to identify and quantify analytes by determining the mass-to-charge ratio (m/z). There are three main steps to MS: Mass-to-charge ratio (m/z) m = molecular weight of the ion z = number of charges present on the measured molecule 2 The ionized sample is broken down into smaller 1 fragments then separated according to m/z ratio. The sample is ionized to give a positive charge. 2 Magnet Sample vaporizer (heater) Sample Magnetic field 1 Detector Gas sample Accelerated ions injection point Electron beam 3 3 The detector quantifies the ions and shows the results as ion peaks relative to the most abundant ion. MS in the biotherapeutic development pipeline MS is used at multiple points during biopharmaceutical development, from the early research stages through to post-approval manufacturing. To further characterize samples, MS is often combined with other analytical tools such as liquid chromatography (LC-MS), capillary electrophoresis (CE-MS). MS can also be combined with imaging using matrix-assisted laser desorption ionization (MALDI) imaging. Structural characterization TECHNIQUE Amino acid sequence profiling MS USE LC-MS can be used to determine the amino acid and terminal amino acid sequences, to characterize the product, compare to the desired product, and investigate batch-to-batch consistency. USED DURING Drug discovery, production for clinical trials, and post approval manufacture. TECHNIQUE Protein structure mapping MS USE LC-MS is used to confirm the product has the appropriate structure, with the correct tertiary structure, such as disulphide bridges. Proteomics MS methods – particularly MS/MS coupling - can use sequential fragmentation to determine structure. USED DURING Drug discovery, production for clinical trials, and post approval manufacture. TECHNIQUE Carbohydrate structure MS USE If the therapeutic is a glycoprotein, carbohydrate content (e.g., glycosylation sites, carbohydrate chains) must be characterized. This can be done using LC-MS, MALDI-MS or CE-MS. USED DURING Production for clinical trials, and post-approval manufacture. Physiochemical and functional properties TECHNIQUE Identifying potential active compounds MS USE One source of biopharmaceuticals are active compounds in plants, microbes and fungi. Ultra high-resolution LC-MS/ MS can be used to rapidly screen large numbers of sources to find natural products and assess their pharmaceutical potential, for example in the discovery of biotin .2 USED DURING Drug discovery TECHNIQUE Molecular weight determination MS USE High-resolution MS is capable of quantifying molecular mass incredibly accurately, making it useful for identifying highly specific biotherapeutic antibodies and differentiating them from similar but clinically irrelevant isotypes. USED DURING Drug discovery TECHNIQUE Analysing drug efficacy MS USE MS techniques can be used to measure downstream effects of target binding, thereby giving insights into its efficiency. For example, MALDI-MS imaging has been used to examine tumor margins, a technique which can be used to assess the efficacy of biotherapeutics for cancer .3 USED DURING Preclinical studies, clinical trials Ensuring quality control and regulatory compliance TECHNIQUE Identifying potential active compounds MS USE The engineered cells producing biotherapeutics also produce a lot of other proteins. If these host cell proteins (HCPs) are included in the final product they can have significant effects on safety and efficacy. LC-MS can identify and quantify these individual contaminating proteins, giving an advantage over other techniques such as ELISA. This allows HCP levels to be monitored during testing for batch release. USED DURING Post-approval manufacture. TECHNIQUE Process-related impurity monitoring MS USE Impurities can accumulate from the manufacturing process itself, including cell media components, processing reagents and solvents. These can also have effects on safety and purity and levels are monitored to be below a certain threshold. LC-MS can rapidly identify and quantify these impurities. USED DURING Post-approval manufacture. TECHNIQUE Post-translational modification (PTM) analysis MS USE As biopharmaceutical products are made in cells, PTMs can occur, such as glycosylated, deamidated or isomerized forms. Again, this can affect safety and efficacy. As many PTMs directly affect the charge of the molecule, LC-MS and CE-MS can be used to assess charge variation and identify the presence of these modifications. USED DURING Post-approval manufacture. TECHNIQUE Mis-formed products MS USE Truncated products formed by enzymatic cleavage or product aggregates can be assessed at the protein level using LC-MS or CE-MS. USED DURING Post-approval manufacture The future of MS in biopharma The complex nature of new therapeutics has significantly increased analytical demands on the biopharmaceutical industry. MS combined with other methods can provide in-depth characterization and precise safety and efficacy data at every stage of biopharmaceutical development and manufacture. However, as these techniques continue to evolve, new roles will emerge for MS in biotherapeutic discovery and development, including : 1 2 3 Automated, online sample The ability to rapidly analyze Coupling with advanced data prep and high-throughput samples in their native state, analysis tools to extract more capabilities to increase for example through ambient meaningful, actionable results efficiency, reduce errors and ionization techniques. from complex MS data. .4 optimize quality control. References 1. Biopharmaceuticals: Large molecules, enormous potential. Boehringer Ingelheim. https://www.boehringer-ingelheim.com/science-innovation/ human-health-innovation/science-stories/biopharmaceuticals-large-molecules-enormous-potential. Accessed February 17, 2025. 2. Montaser R, Kelleher NL. Discovery of the biosynthetic machinery for stravidins, biotin antimetabolites. ACS Chem Bio. 2019;15(5):1134-1140. doi:10.1021/acschembio.9b00890 3. Oppenheimer SR, Mi D, Sanders ME, Caprioli RM. Molecular analysis of tumor margins by MALDI mass spectrometry in renal carcinoma. J Proteome Res. 2010;9(5):2182-2190. doi:10.1021/pr900936z 4. Khalikova M, Jireš J, Horácek O, Douša M, Kucera R, Nováková L. What is the role of current mass spectrometry in pharmaceutical analysis? Mass Spectrom Rev. 2023;43(3):560-609. doi:10.1002/mas.21858