Purification, Quality And Pharmacodynamic Study Of Recombinant Neorudin

A stable and repeatable purification process of recombinant neorudin, a lowbleeding anticoagulant protein, had been developed and can be readily amplified ifonly a minor modification.Thereafter, quality and pharmcodynamic effects of thepurified products were evaluated.The results demonstrated that the quality ofrecombinant neorudin were qualified for clinical study, andrecombinant neorudininhibited markedly the formation of blood clots. The primary results were summed upas follow:（1）The purification process for recombinant neorudin. A molecular weightcut-off of50kD ultrafiltration membrane was selected to remove high molecularweight hybridproteins, from pichia farmentation supernatants. A molecular weightcut-off of2kD ultrafiltration membrane was then selected to concentrate the targetingprotein based on the molecular weight of recombinant neorudin. Thereafter,SepharoseFast Flow and Source15Q chromatography were employed to furtherpurifyrecombinant neorudin. After above mentioned purification process, the purity ofrecombinant neorudin was over95%by HPLC assay, and the recovery was about20~40%. Thus, a purification process for recombinant neorudin was established, andthe reproducibility and stability of the purification process were then confirmed.Moerover, the purification process for recombinant neorudin can be readily amplifiedto improve the purification rate.（2）The quality of recombinant neorudin. The purity detection method ofrecombinant neorudin by HPLC was established after optimization of the elutiongradient, elution rate, sample size, chromatographic column, and the precision, stablility,and reproducibility. The purity of recombinant neorudin by HPLC assay is not less than95%, in other wors, the peak area of recombinant neorudin is not less than95%basedon area normalization method. Real-time PCR method was employed to detect the DNAresidue of pichia, and the standard curve, recovery and precision of the method werethen evaluated. DNA residue of pichia should be less than100pg per dose ofrecombinant neorudin. Protein residue of pichia was examined by ELISA method, andthe standard curve, reproducibility, interference of solvent, and recovery of the methodwere studied as well. Protein residue of pichia should be less than0.05%.（3）Pharmacodynamic effects of recombinant neorudin. The pharmacodynamiceffects of recombinant neorudin were evaluated by the developed model of rabbit arteriovenous shunt thrombosis and rat cerebral infarction. The results showed thatrecombinant neorudin inhibited thrombosis effectively and had less bleeding sideeffects than low molecular weitht hararin and hirudin on the condition of the similareffects, and would provide important references for clinical study.