Protein Aggregation and Performance Optimization Based on Microconformational Changes of Aromatic Hydrophobic Regions.

Protein aggregation is a key concern in biopharmaceutical development and manufacturing. There is growing interest in understanding how the changes in protein microconformation affect the aggregation behavior. This study selected several representative proteins and first manipulated microconformational changes of the aromatic hydrophobic regions of proteins, especially tryptophan residues, by using amine or guanidine additives. The effects of the interactions between the additives and proteins on the aromatic hydrophobic regions could be grouped into three categories: exposure to solvent, burial into core, and no change. The microconformational parameters of the tryptophan residue, including fluorescence peak position (λm), degree of hydrolysis, solvent accessible surface area ( SAS), and packing density ( Den), were obtained by steady-state fluorescence spectroscopy, proteolysis coupled with electrophoresis, and molecular dynamics simulation. Furthermore, the aggregation degrees of globular proteins with distinct surface aromatic hydrophobilities under mechanical stress were investigated. A strong correlation was observed between protein aggregation and the microconformational changes of the aromatic hydrophobic regions incurred by amine or guanidine additives. Protein aggregation was enhanced when the aromatic hydrophobic regions were exposed to the solvent but suppressed when the additives led to burial of the aromatic hydrophobic regions with lower-polarity microenvironment. These findings shed light on the relationship between protein aggregation and molecular conformation and paved way for future preformulation studies of therapeutic proteins.

Damage of proteins at the air/water interface: Surface tension characterizes globulin interface stability.

In the present study, we aimed to see what circumstances may cause protein damage at air/water interface and reveal the correlation between the surface properties of protein solution and the interface stability. The surface hydrophobicity and ß-sheet of protein were determined by exogenous fluorescent probes, and the changes in the spatial structure of proteins were characterized by steady-state fluorescence spectroscopy. The surface tension was determined by the plate method, and such value was used to establish the correlation with the hydrophobicity and structure of the protein. Moreover, degree of aggregation in the presence or absence of Hofmeister salt in protein solution was investigated. There was a significant correlation between the surface tension and hydrophobicity of the protein solution (P < 0.05). The surface tension and structure of the protein also showed a significant correlation under the induction of pH (P < 0.05). Furthermore, when the protein was induced by the air/water interface, the surface tension, hydrophobicity, and structure of proteins were correlated, and protein aggregation was increased. When the additive induced a decrease in the surface tension of the protein solution, the protein aggregation was promoted. These findings provided valuable insights into the relationship between surface tension of the protein solution and interfacial stability and paved the way for future pre-formulation studies of therapeutic proteins.