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Biochemistry

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

Published: June 16th, 2023

DOI:

10.3791/65298

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....

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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.......

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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.......

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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.......

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The authors thank Robert Schuster for assistance with data verification.

....

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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 https://proteinmetrics.com/byos/
Version 4.5
GPMAW GPMAW http://www.gpmaw.com/
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

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