Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint () by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.