Separation, Purification And PEGylation Of Hirudin

Hirudin is the most potent inhibitor of thrombin found in nature.The extraction, purification and PEGylation process of it was studied in this paper,and the modified products' in vitro activity and apparent molecular weight was determined.A lot of researches on the isolation of hirudin have been carried out in recent years.Three stages of pretreatment,extraction and purificaiton for hirudin were studied in this paper. Microwave treatment,buffer extraction,drying and freeze drying methods were tested and compared in the pretreatment of leech.In the extraction procedure,pH selective methods was proved to be the best method to conserve the activity in vitro,3.19 ATU/mg.Anion exchange and gel filtration were used to purify the crude extract.The active fraction conserved 9.1×10~3 ATU/mg activity in vitro after the anion exchange procedure.Hirudin was proved to be highly purified by SDS-PAGE analysis.Although hirudin has the strongest anti-thrombin activity,its short half-life in serum, significantly limits its clinical anticoagulant application.Currently,PEGylation is commonly used as an effective method to prolong its half-life in serum.But there are some short comings in it:the complexity of the modified products and the reduced bioactivity after PEGylation.To solve these problems,a novel method assisted by anion exchange column which achieved the advantage of reaction and separation in a single unit was adopted to PEGylate recombinant hirudin 2(ab.HV2) using SC-PEG 5 kDa and SC-PEG 20 kDa respectively in the paper.PEGylation using SC-PEG 20 kDa would inhibit over-PEGylated products. However,the conversion ratio decreased compared with that modified with SC-PEG 5 kDa. On-column modification could also promote the homogeneity of the products.The optimal reaction conditions for HV2 modified with SC-PEG 20 kDa on column were investigated.If the PEGylated reaction was conducted for 1 hour in the PBS at pH 8.0 in a mPEG/HV2 molar ratio of 9:1,a higher yield would be achieved.Investigation of the specific activity in vitro of mono-PEGylated HV2 shows that mono-PEGylated HV2 prepared on column conserved much more specific activity than that prepared in solution phase,while PEGylated 20 kDa HV2 conserved specific activity better than PEGylated 5 kDa HV2.The modified site of hirudin might be K35 by molecular dynamics simulation.Gel filtration was used to test the stability of the modified products.The multi-modified products are not stable.They would degrade to PEGylated hirudin in lower modification extent.The apparent molecular weight of the modified products was 4.4～6.2 times of the conjutated PEG molecular weight.