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.

NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF.

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity.