Studies on site-specific PEGylation of uricase from Bacillus fastidious using mPEG-derivatives

Abstract

Uricase (Urate oxidase) is a therapeutic enzyme which is administered for the treatment of hyperuricemic and gout patients. Uricases are considered supreme and most effective in the treatment of refractory gout (Yang et al. 2012). It is presently administered in its randomly conjugated/PEGylated form as Uricase-Polyethylene glycol (PEG) conjugate, however, it suffers from significant efficacy-related shortcomings. The present randomly PEGylated uricase formulations available in the market are Rasburicase and Pegloticase. Rasburicase ((Fasturtec ® / Elitek ® ) (a recombinant uricase from Aspergillus flavus) has a monthly dose of 10 mg/kg body weight and elicited an immune response (Vogt, 2005). Pegloticase (Krystexxa ® ) (recombinant mammalian uricase modified with methoxy-PEG) has a biweekly dosage of 0.14 mg/kg body weight (Sclesinger, 2011) and elicited an immune response against mPEG (Yue et al. 2008; Ganson et al. 2006). Considering a very interesting case of site-specific PEGylated Interferon α-a, only 4 μg of weekly dosage was sufficient for the management of chronic Hepatitis C with reduced immunogenicity (Foster, 2010; Rodriguez-Torres et al. 2009). Hence, site- specific PEGylated uricase can prove to be an efficient alternate PEG therapeutic to overcome the demerits of the existing uricase therapeutics. The present work encompassed the development of site-specific PEGylated uricase conjugates following the implementation of two different second generation PEGylation strategies namely thiol and N-terminal PEGylation. mPEG-maleimide and mPEG-propionaldehyde were used as PEGylating reagents for thiol and N-terminal PEGylation respectively. The PEGylation reaction conditions which influenced the yield of the site-specific uricase conjugates were optimized to achieve a higher conjugate yield and productivity. The uricase conjugates obtained were purified using ultrafiltration, gel filtration chromatography and size exclusion fast protein liquid chromatography (SE- FPLC). The purified uricase conjugates were characterized by their residual activity, the degree of modification, molecular weight, size, conformational changes, storage stability, kinetic and immunological properties. iThis is the first report on synthesis of site-specific uricase conjugates and optimization of PEGylation reaction conditions for their production. The PEGylated uricase conjugates obtained by both thiol and N-terminal PEGylation strategies possessed better storage stability and residual activities in comparison to the residual activities possessed by PEGylated uricase conjugates reported till date. The site-specific uricase conjugates also displayed a 60-70 % reduction in immunogenicity compared to native uricase. The conjugates synthesized in the present study appeared to have beneficial and long-acting uricolytic effects for curing hyperuricemia and gout in comparison to the random PEGylated uricase. This site-specific uricase can be a potential conjugate for further studies related to characterization and immunological studies.