Removal Of Endotoxin From Medicine Preparations By New Designed Affinity Membrane Cartridges And Approach To Determine The Sensitivity Of Tachypleus Amebocyte Lysate

Affinity membrane chromatography based on the specific interaction between ligand immobilized on matrix and its ligand has been paid close attention to be used for biomolecules separation and purification.Endotoxin (ET)is a main contaminant in parenteral medicines and the removal of it from medicines, especially genie recombination proteins, has been the target for researches and productions.Both dye ligands and chelate metal ions ligands were immobilized onto acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorptions of the prepared matrixes for either bovine serum albumin (BSA)or human serum albumin (HSA) were investigated and proved to follow the Freundlich equation. In static adsorption of human plasma, the adsorption capacity of HSA were 12.5 mg/g fiber and one band was observed in SDS-PAGE electrophoresis upon the filter rod immobilized with Cibacron Blue F3GA. Ligand utilization efficiencies and breakthrough volumes of HSA adsorption when the feed-rates were from 0.5 to 6 mL/min by the Cibacron Blue F3GA immobilized filter rod were obtained. In the affinity chromatography of human plasma, the yield of human serum albumin was 1.27g per 100mL plasma.Three affinity membrane cartridges were designed as follows: a membrane cartridge for endotoxin removal in high flux, a modified membrane cartridge construction and its preparation, a simple membrane cartridge construction and its preparation. Besides, an apparatus for making membrane cartridges was invented for membrane cartridge production. Also the manufacturer's standard for making the membrane cartridge was established.The effects of nine salts on endotoxin determination in buffer and medicines were investigated. As a result, 250mM pH7.5-8 Tris-HCl buffer was chosen as a dilution for samples containing high concentration salts in medicines and in the intermediate product. By compared the difference between the standard curves of different sensitivity of Tachypleus amebocyte lysate (TAL), methods to determine,thesensitivity of LAL were raised based on recovery and reactive time in endotoxin determination.The suitability of affinity membranes and their cartridges to be used for the removal of endotoxin from distilled water, phosphate buffer solution, human serum albumin solution, interferon preparations, creatine phosphate, influenza vaccine, ascites sample and sodium alginate solution was examined. For example, the endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml respectively when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH3.9 was applied to the chitosan immobilized membrane cartridge at a flow rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. For an influenza vaccine sample, the endotoxin content was reduced from 400 EU/ml to less than 100 EU/ml while the protein recovery was 88%.