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.

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 N-H 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.