Study On Uricase Modification With PEG-PSA Block Co-polymer

Natural and recombinant proteins, peptide drugs play an increasingly important role inthe treatment of incurable diseases, but its efficacy was greatly discounted due to its poorstability, short half-life, immunogenicity. Polyethylene glycol modification has significantlyenhanced proteolytic stability, prolonged circulation half-life and reduced immunogenicity,Unfortunately some patients poduced antibodies against PEG in the chronic treatment due toaccumulation of high molecular weight polyethylene glycol in the body. In this paper, uricasewas modified with block polymer, of which biodegradable polysialic acid was linked withbifunctional PEG, to reduce its antigenicity and play a major role in chronic therapy withexpectation.The crude extract polysialic acid was refined by anion exchange chromatography. Thecollected target peak was determined for polysialic acid and protein content, and thepolysialic acid recovery ratio was79.7%, which was relativly high. While protein content wasreduced from1.99×10-3mg protein／mg PSA to9.16×10-4mg protein／mg PSA.Thereafter the sample was desalted by ultrafiltration and lyophilized. The purified PSA wasactivated following two steps: firstly, the aldehyde group was generated by periodateoxidation at the non-reducing end, and then the active thiol group was formed usingcystamine and dithiothreitol. Aldehyde activation was100%detected by DNPH method, andthe thiol group activation was38.6%±2.68%by Ellman method.Activated polysialic acid reacted with hetero-bifunctional polyethylene glycol to form ablock co-polymer at25°C for half an hour, which modified uricase at4°C thereafter. Theconjugate was purified with gel permeation chromatography (TOSOH Toyopearl HW-55F).The related peaks was collected and identified by chemical method, the first effluent peak hadthe highest content of polysialic acid, polyethylene glycol and protein among four peaks,suggesting the success of block polymerzation modification. Changes in the molecular weightof the conjugate was identified by SDS-PAGE, which displayed45and55kDa fuzzy bandscompared to the35kDa band of native enzyme.The molecular weight of the conjugate was521.4kDa through multi-angle laser lightscattering gel system, while the MW of uricase, polysialc acid, PEG showed140kDa,34kDa,and3.5kDa, respectively, thus each tetramer linked10.08modifiers according to molecularweight information, each monomer connected2.52modifiers. Compared to native enzyme,residual activity of modified enzyme was72.4%, and heat inactivation half-life in vitro wasimproved from115.5h up to231h. The cross-linking ratio was12.3, amino modification ratewas10.6%. The tolerance stability from heat, acid and alkaline, trypsin treatment wassignificantly improved.Other modification methods were applied to uricase modification. For one approach,reductive amination modification of uricase was employed, namely hydroxyl groups at thenon-reducing end of polysialic acid were oxidated to form the aldehyde group, which reactedwith amino group of uricase at25°C for48h to form conjugate in the present of reducingagent. For another, PEGylation of uricase was applied at4°C for2h to form conjugate. The modification products were purified with gel filtration chromatography, indetified bychemical method and SDS-PAGE.Comparison of three approaches of modification, PEGylation modification has thehighest extent of modification, the cross-linking ratio was18.10and amino modification ratewas14.1%, follewed by block polymerization modification, polysialation modification hasthe minimum degree of modification. Block polymerization modification showed the highestenzyme residual activity of72.4%relative to native uricase, PEGylation modification got53.6%with the lowest enzyme residual activity; PEGylation modification had the longest half-life upto402h, half-life of block polymerization and polysialylation modification was close. Theantigenicity of three approaches of modification decreased compared to the unmodifieduricase, copolymerization and reductive amination modification had similar antigenicity,while PEGylation modification was relatively higher.