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The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass‐spectrometry‐based workflow that allows rapid and facile molecular characterization of antibody‐based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time‐resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre‐clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance‐derived kinetic and equilibrium binding parameters.

The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen− deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.

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