Hybrid Mass Spectrometry\nApplied across the Production\nof Antibody Biotherapeutics

Post expression from the host cells, biotherapeutics\nundergo downstream\nprocessing steps before final formulation. Mass spectrometry and biophysical\ncharacterization methods are valuable for examining conformational\nand stoichiometric changes at these stages, although typically not\nused in biomanufacturing, where stability is assessed via bulk property\nstudies. Here we apply hybrid MS methods to understand how solution\ncondition changes impact the structural integrity of a biopharmaceutical\nacross the processing pipeline. As an exemplar product, we use the\nmodel IgG1 antibody, mAb4. Flexibility, stability, aggregation propensity,\nand bulk properties are evaluated in relation to perfusion media,\npurification stages, and formulation solutions. Comparisons with Herceptin,\nan extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible\nsolution. Despite presenting similar charge state distributions (CSD)\nin native MS, mAb4, and Herceptin show distinct unfolding patterns\nin activated ion mobility mass spectrometry (aIM-MS) and differential\nscanning fluorimetry (DSF). Herceptin’s greater structural\nstability and aggregation onset temperature (<i>T</i><sub>agg</sub>) are attributed to heavier glycosylation and kappa-class\nlight chains, unlike the lambda-class light chains in mAb4. Hydrogen–deuterium\nexchange mass spectrometry (HDX-MS) revealed that mAb4 undergoes substantial\nstructural changes during purification, marked by high flexibility,\nlow melting temperature (Tm), and prevalent repulsive protein–protein\ninteractions but transitions to a compact and stable structure in\nhigh-salt and formulated environments. Notably, in formulation, the\nthird constant domain (CH3) of the heavy chain retains flexibility\nand is a region of interest for aggregation. Future work could translate\nfeatures of interest from comprehensive studies like this to targeted\napproaches that could be utilized early in the development stage to\naid in decision-making regarding targeted mutations or to guide the\ndesign space of bioprocesses and formulation choices.