Lysine-PEGylation of Moroctocog Alfa: Purification and Activity Analysis
Ahmad Hashemzehi1, Alireza Vatanara1, Loghman Firoozpour2
1Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Introduction:
Hemophilia A is characterized by a deficiency in the activity of coagulation Factor VIII (FVIII), with the severity of the disease determined by the patient's baseline plasma FVIII levels. FVIII is predominantly synthesized in the liver and undergoes intracellular transport facilitated by chaperone proteins. PEGylation of FVIII has been demonstrated to extend its half-life and reduce immunogenicity.
Materials And Methods:
A solution of Moroctocog alfa, derived from the Safacto manufacturing process, was adjusted to a pH of 7.4. Polyethylene glycol (PEG) with a molecular weight of 5,000 Da (PEG-SS5000) was then added to the solution under gentle stirring. Fast protein liquid chromatography (FPLC) was employed to separate PEGylated Moroctocog alfa from the non-PEGylated form. The conjugation of methoxy PEG succinimidyl succinate to Moroctocog alfa was analyzed using SDS-PAGE. The activity of Moroctocog alfa was assessed using an STA compact analyzer, which measures clot formation through electromagnetic detection.
Results:
SDS-PAGE confirmed the presence of both unmodified and PEGylated forms of Moroctocog alfa. The activity analysis revealed that PEGylated Moroctocog alfa exhibited a reduced activity level compared to its non-PEGylated counterpart.
