Effects of sucrose on rFVIIa aggregation and methionine oxidation.

The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from the protein's surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon incubation. Unlike aggregation, methionine oxidation was found to increase in the presence of sucrose. The two methionine residues susceptible to oxidation are presumably located at the protein surface, and the chemical potential increase in the presence of sucrose may account for the increase in oxidation rate. While FTIR spectroscopy suggested that sucrose induces small conformational changes in the rFVIIa structure, CD spectroscopy did not support this finding. The secondary structure of precipitated rFVIIa was changed when compared to the native solution secondary structure. Appearance of bands characteristic of intermolecular beta-sheet structure were found coincident with a decrease in alpha-helix and intramolecular beta-sheet structure.