A Decoupled Automation Platform for Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass-spectrometry-based approach.

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.