Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

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 (Tm) 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 Tm 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.

HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions.

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF.