Study On Preparation Process And Quality Standard Of Difibrase

According to its biological character, snake venom of Agkistrodonhalys wasidentified by different methods. In this paper, the separation and purification process andquality standardof difibras from snake venom of Agkistrodonhalys were studied.The snake venom was separated through ion exchange media sourse 15Q, sourse15S and sourse 30S. The active elute was retained and most other proteins were removed.The sourse 30S was cheaper and higher resolution in low-pressure system. The elute fromion exchange was subjected to affinity chromatography with mediaHeparin-sepharoseCL-4B, Heparin-sepharoseCL-6B and Heparin-sepharose6 Fast Flow,reapetively. The three affinity maps were almost the same, Heparin-SepharoseCL-6B waschosed as cost effective. The active elute was collected as crude difibrase, and separatedby hydrophobic chromatography using three different media Sourse 15PHE, Sourse15ETH and Sourse 15ISO. All of the three hydrophobic maps showed two peaks, and thesecond peak had the activity. Sourse 15PHE resulted in the difibrase activity higher thanthe other two media. The active elute was separated by Superdex 200, Superdex 30 andSuperdex 75 as gel chromatography media, respectively. Each of gel chromatographygave the similar result with only one eluting peak. Superdex 75 was selected as mediumfor its high adsorption, recovery rate and selectivity.After selection the media and parameters in in the lab-scale, the process was furtherscaled up. The preparation process was as following: The snake venom was firstseparated with ion exchange followed by affinity chromatograph, filtered the componentto removal the microbes, then subjected to hydrophobic chromatography andultrafiltration, finally to Superdex 75 for gel chromatography. The purity of the activitypeak was higher more than 99.0% in analysis by HPLC. The SDS-PAGE showed onlyone band with the molecular weight of 36000. This separation and purification processwas applicable to practice with simple operation and removable to neurotoxin,haemorrhage toxin and other toxin. Compared with other products, the snake difibrasehad higher purity, lower side effect and more stable quality.According to the China Pharmacopeia 2000, the physical, chemical and biologicalcharacters of the difibrase were analyzed and detected. The results showed that thedifibrase was stabile in 36 mothons at room temperature and the expiration period was intwo years. The standard of quality control was established. Compared with t-PA,urokinase and streptokinase, the snake venom difibrase has its own properties, and is apromising thrombolysis drug as substitute suppository or combination with otherthrombolysis drug in the clinic use.