Protective role of l-tyrosine in the sterilization of Ceruloplasmin therapeutic protein by gamma-irradiation

Abstract

Gamma-irradiation of proteins in aqueous media at doses higher than 5 kGy, is known to induce their aggregation (without oxygen) or degradation (in presence of oxygen). The effect of l-tyrosine (Tyr) addition before irradiation of therapeutic proteins, i.e. ceruloplasmin (CP), was examined. It was found that the presence of l-tyrosine during irradiation in oxygen free conditions prevents the protein aggregation even at doses as high as 10 kGy, probably by scavenging oxygen radicals produced by irradiation. The protective role of l-tyrosine allowing the gamma-irradiation treatment of therapeutic proteins in solution, without conformational alteration was asserted by Sodium Dodecyl Sulphate—Polyacrylamide Gel Electrophoresis and by High Performance Size Exclusion Chromatography. Total viable microorganisms content evaluated by Plate Count Agar incubation for 48 h at 30°C, showed that CP protein preparations gamma-irradiated in presence and absence of l-tyrosine remained sterile and maintained its enzyme activity during at least eight weeks of storage at 4°C. At day zero of irradiation, the sterility was reached in all variants (CP, CP+Tyr, Tyr) at doses higher than 4 kGy. However, during storage, sterility was also found for 3 kGy irradiated CP and for 2 kGy irradiated CP+Tyr.