Study On The MonoPEGylated Recombinant Hirudin

Hirudin, the most potent inhibitor ofthrombin found in nature, is of short half-life inserum, which significantly limits its clinical application. Recently, PEGylation iscommonly used as an effective method to prolong the half-life of protein and peptidedrugs in serum. Many PEGylated protein drugs have come into the market, likePEG-Intron (PEGinterferonα-2b), PEGasys (PEGinterferonα-2a), Neulasta (PEG G-CSF)etc. The PEGylation has greatly improved the half-life in vivo and the bioavailability ofthese drugs. Researches in the area of PEGylation of recombinant hirudin(r-Hirudin)commonly concentrate on the the lysine residue PEGylation under basic conditions, thiswill inevitably lead to the active center(Lys47) PEGylation, and the reaction products areususlly a mixture of quadri-, tri-, di-, mono-PEGylated species. In order to avoid thePEGylation of active center and obtain homogeneous monoPEGylated product, twostrategies were applied in this paper.First, the mild acidic PEGylation strategy was used to target the only histidineresidue(His51) of r-Hirudin. Succinimidyl carbonyl mPEG(SC-mPEG, 20kDa) wasattached to r-Hirudin at pH 6.0, the reaction mixture was easily separated by a one-stepion-exchange chromatographic(IEC) procedure, and 94.6％of the PEGylation productwas monoPEGylated. About 79.71％of the monoPEGylated r-Hirudin was histidinepositional isomer, which showed the acidic PEGylation operation avoided the PEGylationof Lys47 in principle. The more than 95％pure predominant monoPEGylated productwas obtained and retained 34％of the anticoagulant activity in vitro.In adition, IEC was used to assisted the PEGylation of r-Hirudin. The reaction wasconducted under the reaction condition of pH 6.0, 20mM PBS, r-Hir:SC-mPEG=1:3, andthe PEGylation and seperation were conducted in one step. Only monoPEGylated productwas shown in SDS-PAGE, and the PEGylation wasn't occurred at His51. According tothe PEGylation reaction theory, it may occurr at the N-terminal amine group. The purityof monoPEGylated product was more than 95％, and 34.85％of the anticoagulant activitywas retained in vitro.