Abstract
High concentration biotherapeutic formulations are often required to deliver large doses of drugs to achieve a desired degree of efficacy and less frequent dose. However, highly concentrated protein-containing solutions may exhibit undesirable therapeutic properties, such as increased viscosity, aggregation, and phase separation that can affect drug efficacy and raise safety issues. The characterization of high concentration protein formulations is a critical yet challenging analytical task for therapeutic development efforts, due to the lack of technologies capable of making accurate measurements under such conditions. To address this issue, we developed a novel dilution-free hydrogen/deuterium exchange (HDX) mass spectrometry (MS) method for the direct conformational analysis of high concentration biotherapeutics. Here, we particularly focused on studying phase separation phenomenon that can occur at high protein concentrations. First, two aliquots of monoclonal antibodies (mAbs) were dialyzed in either hydrogen- or deuterium-containing buffers at low salt and pH. Phases that separated were then discretely sampled and subjected to dilution-free HDX-MS analysis through mixing the non-deuterated and deuterated protein aliquots. Our HDX-MS results analyzed at a global protein level reveal less deuterium incorporation for the protein-enriched phase compared to the protein-depleted phase present in high concentration formulations. A peptide level analysis further confirmed these observed differences, and a detailed statistical analysis provided direct information surrounding the details of the conformational changes observed. Based on our HDX-MS results, we propose possible structures for the self-associated mAbs present at high concentrations. Our new method can potentially provide useful insights into the unusual behavior of therapeutic proteins in high concentration formulations, aiding their development.