JoVE Logo
Faculty Resource Center

Sign In





Representative Results






An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules

Published: June 16th, 2023



1GlaxoSmithKline, 2Merck, 3Moderna

This article describes the use of a software application, mAbScale, for the calculation of masses for monoclonal antibody-based protein therapeutics.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers.

We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.

Over the past two decades, biotherapeutics have evolved to become a mainstay of the modern pharmaceutical industry. The SARS-CoV2 pandemic and other life-threatening conditions have further increased the need for the faster and broader development of biopharmaceutical molecules1,2,3.

The biotherapeutic molecular weight is critical for the identification of the molecule, in combination with other analytical assays. The intact and reduced subunit masses are used throughout the discovery and development lifecycles as part of cont....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The high-level workflow for mAbScale is shown in Figure 2. Each step has more sophisticated inner decision branches, loops, and combinatorics. A detailed algorithmic workflow describing the calculation process is presented in Supplementary Figure 1. The application output is saved in a spreadsheet format in the user-selected folder. The output file consists of multiple separate worksheets, which can be categorized as the user input, molecular weight calculations, and referen.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

A variety of mAbs were selected to represent different types of mAbs. A commercially available mAb standard was selected to represent a conventional mAb with identical heavy chains, identical light chains, and one N-linked glycosylation site in the Fc region. A mAb with an additional light chain N-linked glycosylation, a bispecific mAb, and an antibody-drug conjugate (ADC) mAb were also chosen to widen the application usage. The chemical composition, calculated mass, measured mass, and mass error of these example mA.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

mAbScale provides an intuitive user interface with the flexibility to alter the building blocks for mass and elemental calculations. The users are expected to have a basic understanding of the target molecule to use the application, derive correct masses, and interpret the results. For example, the intact or reduced mass output sheet can be overwhelming due to the numerous rows of intact or reduced masses, since the default glycan database contains 88 N-linked glycans that are commonly found in the Fc portion of therapeu.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors thank Robert Schuster for assistance with data verification.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Acquity UPLC system  Waters Corp., Milford, MA N/A Modular system
Antibody-drug conjugate (ADC) GlaxoSmithKline N/A Proprietory molecule
BEH 200 SEC column  Waters Corp., Milford, MA 176003904
Bispecific mAb GlaxoSmithKline N/A Proprietory molecule
Byos Protein Metrics, Cupertino, CA
Version 4.5
LC-MS grade water  Thermo Fisher Scientific, Waltham, MA W6-1
mAb standard  Waters Corp., Milford, MA 186009125 Waters Humanized mAb Mass Check Standard
mAbScale GlaxoSmithKline Apache License, Version 2.0 
Xevo G2 Q-TOF mass spectrometer Waters Corp., Milford, MA N/A Modular system

  1. Reichert, J. M., Valge-Archer, V. E. Development trends for monoclonal antibody cancer therapeutics. Nature Reviews Drug Discovery. 6 (5), 349-356 (2007).
  2. Kintzing, J. R., Filsinger Interrante, M. V., Cochran, J. R. Emerging strategies for developing next-generation protein therapeutics for cancer treatment. Trends in Pharmacological Sciences. 37 (12), 993-1008 (2016).
  3. Wang, M. -. Y., et al. SARS-CoV-2: Structure, biology, and structure-based therapeutics development. Frontiers in Cellular and Infection Microbiology. 10, 587269 (2020).
  4. ICH Q8 (R2) Pharmaceutical Development - Scientific Guideline. European Medicines Agency Available from: (2018)
  5. Donnelly, D. P., et al. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nature Methods. 16 (7), 587-594 (2019).
  6. Gadgil, H. S., Pipes, G. D., Dillon, T. M., Treuheit, M. J., Bondarenko, P. V. Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies. Journal of the American Society for Mass Spectrometry. 17 (6), 867-872 (2006).
  7. Beck, A., Sanglier-Cianférani, S., Van Dorsselaer, A. Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. Analytical Chemistry. 84 (11), 4637-4646 (2012).
  8. Camperi, J., Goyon, A., Guillarme, D., Zhang, K., Stella, C. Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches. Analyst. 146 (3), 747-769 (2021).
  9. Liu, H., May, K. Disulfide bond structures of IgG molecules. mAbs. 4 (1), 17-23 (2012).
  10. Jakes, C., Füssl, F., Zaborowska, I., Bones, J. Rapid analysis of biotherapeutics using protein a chromatography coupled to orbitrap mass spectrometry. Analytical Chemistry. 93 (40), 13505-13512 (2021).
  11. Robotham, A. C., Kelly, J. F., Matte, A. Chapter 1 - LC-MS characterization of antibody-based therapeutics: Recent highlights and future prospects. Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. , 1-33 (2020).
  12. Valeja, S. G., et al. Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry. 83 (22), 8391-8395 (2011).
  13. Fornelli, L., Ayoub, D., Aizikov, K., Beck, A., Tsybin, Y. O. Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry. Analytical Chemistry. 86 (6), 3005-3012 (2014).
  14. Berglund, M., Wieser, M. E. Isotopic compositions of the elements 2009 (IUPAC Technical Report). Pure and Applied Chemistry. 83 (2), 397-410 (2011).
  15. Wang, M., et al. The Ame2012 atomic mass evaluation. Chinese Physics C. 36 (12), 1603-2014 (2012).
  16. Peri, S., Steen, H., Pandey, A. GPMAW--A software tool for analyzing proteins and peptides. Trends in Biochemical Sciences. 26 (11), 687-689 (2001).
  17. Tipton, J. D., et al. Analysis of intact protein isoforms by mass spectrometry. The Journal of Biological Chemistry. 286 (29), 25451-25458 (2011).
  18. De Leoz, M. L. A., et al. interlaboratory study on glycosylation analysis of monoclonal antibodies: Comparison of results from diverse analytical methods. Molecular & Cellular Proteomics. 19 (1), 11-30 (2020).
  19. Cymer, F., Beck, H., Rohde, A., Reusch, D. Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential. Biologicals. 52, 1-11 (2018).
  20. Baker, P. R., Trinidad, J. C., Chalkley, R. J. Modification site localization scoring integrated into a search engine. Molecular & Cellular Proteomics. 10 (7), (2011).
  21. Chalkley, R. J., Clauser, K. R. Modification site localization scoring: Strategies and performance. Molecular & Cellular Proteomics. 11 (5), 3-14 (2012).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved