Pegylation Of Recombinant Uricase From Bacillus Fastidiosus And Evaluation Of MPEG₅k Modified Uricase

Recombinant uricase from Bacillus fastidiosus (ATCC29604) expressed in E. coli was purified by DEAE-cellulose chromatography twice with the specific activity of 6.0 IU/mg. Preliminary pharmacodynamics results indicated a very short circulating time of the native uricase in rats (approximately 10¹5min) and strong immunogenicity. Pegylation of protein is one of the most efficient strategies for enhancing medicinal properties of protein drugs. The aims of our work were focus on optimizing the processes of pegylation of uricase and elementarily evaluating the medicinal properties of mPEG5k modified uricase.1 Preparation of mPEG derivativesBased on the structural characterization of uricase produced in our lab, we prepared several modification reagents aiming to amine developed from mPEG by two ways. One strategy was that mPEG firstly reacted to succinic anhydride in THF solvent, and consequently dehydrated together with NHS with the aid of DCC, generated SS-mPEG5k and SS-mPEG₃50 with a yield of 60⁷5%, 56%, respectively. Alternatively, mPEG was initially reacted with triphosgene and then transited into SC-mPEG5k, yielded 87%. Beside, acetic acid directly reacted to NHS with the presence of dehydration of DCC, and yielded 90% of Ac-NHS. All products were identified by TLC.2 Optimization of the processes of modification of uricaseTo optimize the modification of uricase, three factors were investigated:the quantities of modification reagents, size of molecule of modification reagents and effect of ligands, respectively. To start with, when molar ratio (modification reagent : uricase) increased from 0 to 100, there was a linear relationship between the degree of modification of uricase and the quantities of modification reagents (y=0.67141x-4.9586, R2=0.9972). At molar ratio of 200, the degree of modification of uricase reached to 80%. The maximum degree of modification reached to 85% when molar ratio of the modification reagent to uricase increased to 500. But the retention of activity of uricase displayed a persistent decline with a minimum of 17% of native activity. Moreover, the length of mPEG chain was taken into account in the process of our work. We compared the processes of uricase modified by three kinds of modification reagents differing in size of molecule, SS-mPEG5k, SS-mPEG350 and Ac-NHS, separately. The maximum modified degree of uricase was 75%, 86% and 94%, respectively (measured by TNBS) and the minimum retention of activity of PEGylated uricase was 60-70%, 23% and approximately 10%, respectively. The last factor was the effect of ligands to the residual activity of SC-mPEG5k modified uricase with 200 molar ratio of modification reagent to uricase. Uricase linked to mPEG without the protection of ligands always showed a dramatically loss of native activity, approximately 20-30% of native activity in the final conjugation. Amazingly, at similar degree of modification, uricase modified with the presence of ligands of uricase, 30μM of oxonic acid, 150μM of xanthine and initial 150μM of uric acid, separately, displayed a notable improvement of retention of activity, reaching to 70%, 59%, and 53%, respectively.3 Evaluation of the druggability of mPEG5k modified uricaseNative and mPEG modified uricase have similar responses to change of pH value and effect of cations like Mg2+. In the case of the thermal stability and the capability of resistance to protease hydrolysis, compared with native, pegylated uricase demonstrated a remarkable enhancement. To evaluate the pharmacokinetics and pharmacodynamics profiles of native and mPEG5k modified uricase, we established high-level uric acid SD rats by raperitoneal injection of 0.20mg/g of oxonate potassium, a strong inhibitor of uricase (Ki≦3μM), per day for 4-6 weeks. The mean level of plasma uric acid excessed 0.42mM, the value of clinical diagnostic criteria of hyperuricemia. However, the animal models were not suitable to be used to evaluate the properties of uricase as a result of high-level of inhibitor of uricase which was crucial to maintain the high-level of plasma uric acid through inhibiting the catalytic activity of endogenous uricase, but simultaneously brought out intense inhibition to the active of both native and pegylated uricase. Hence, we devised a mimetic strategy that combined the capability of lowering uric acid in vitro and the pharmacodynamics of native and mPEG modified uricase in vivo to appraise the druggability of enzymatic preparations. Both native and modified uricase exhibited a rapid capability of lowering uric acid. Strikingly, compared to a swift elimination of native uricase whose half-life of plasma uricase activity was approximately 12min, mPEG5k modified uricase show a conspicuous elongation, approximately 20h in rats. In addition to those strategies above mentioned, avian was also employed to evaluate the medicinal properties of native and mPEG5k modified uricase in virtue of lacking of endogenous activity of uricase. The results revealed that mPEG5k modified uricase possessed a longer retention of residual activity in vivo and a preferable capability of maintaining a lower level of plasma uric acid. Besides, in the experiment of immunological properties of native and mPEG5k modified uricase, the data indicated that native uricase show strong immunogenicity with extremely high titer of anti-uricase IgG antibody in rabbits (1:781250); mPEG5k modified uricsae displayed a relatively low immunogenicity which was as low as 5% of native uricase in rats. Alsoly, anti-mPEG5k IgM antibody was observed in rats developed by mPEG5k modified uricase. Besides, the capability of mPEG5k modified uricase for binding to rabbit anti-uricase IgG antibody was low as 10% with comparison of native uricase.According to these works, it could be concluded that the procedure of modification of uricase was affected by the size and quantities of modification reagent. Besides, the ligands of uricase, like xanthine and oxonic acid could provide a protection for the catalytic sites and lead to higher retention of activity in the final conjugation. The mPEG5k modified uricase showed better medicinal properties compared to the native.