The effect of glycerol and protein structure on chemical degradation through deamidation and isomerization

Therapeutic proteins are essential in modern medicine but are often susceptible to chemical degradation during production, transportation, and storage, which can compromise functionality and even lead to immunogenicity in patients. This study investigates how the structure of the affibody GA-Z affects its chemical stability and how glycerol stabilizes the protein against deamidation and isomerization. Degradation of individual residues in GA-Z was quantified using Liquid Chromatography-Mass Spectrometry in the presence and absence of glycerol, while glycerol-induced conformational changes were followed using an AlphaFold2 structure of GA-Z and two-dimensional Nuclear Magnetic Resonance spectroscopy. Thermal stability was assessed by Differential Scanning Calorimetry and Differential Scanning Fluorimetry. The results reveal that the stability of the residues depends on local structures and that the effect of glycerol depends on the residue type and local structure. Glycerol destabilized aspartic acid residues in unstructured regions but significantly stabilized all residues in ordered regions. For these residues, a higher initial degradation correlated with stronger stabilization, independent of the residue type and the structural motifs for degradation. The structural analysis shows that glycerol enhances GA-Z stability by stabilizing the folded state of the protein and by increasing the α-helical population. Overall, this research contributes to a deeper understanding of protein stability and the stabilizing effect of glycerol.