Study On Endotoxin Removal From Biological Products By PMB Affinity Adsorbent

Many biological products are produced by the Gram-negative bacteria. These products are always contaminated with endotoxin. The removal of endotoxin is essential for a safe parenteral administration. Pertussis vaccine is prepared by Bordetella pertussis, and the endotoxin concentration of the stock solution is very high. The removal efficiency of entotoxin in ion-exchange chromatography (IEC) is above 95%, however, the residual endotoxin concentration is still very high, which must be removed. Polymyxin B (PMB) affinity adsorbent was synthesized and used to remove endotoxin from the biological products. The effect of medium property, solution conditions, protien property and operation conditions on endotoxin removal and protein recovery was studied, and then an efficient process for endotoxin removal was developed.Firstly, PMB was coupled to epoxy-activated agarose to prepare PMB QZT FF affinity adsorbent. The effect of the epoxy activation agents, pH value and the quantity of PMB added on the sythesis of the adsorbent was investigated, and then a method for preparing PMB affinity adsorbent with adjustable space arm and ligand density was established.Furthermore, the PMB affinity adsorbent prepared above was used to remove endotoxin from different protein solutions and the effect of medium property, solution conditions, protein property and operation conditions on endotoxin removal and protein recovery was studied. The results indicated that the ligand density, pH value, salt concentration and protein property all have remarkable effects on endotoxin removal. The electrostatic interaction between the molecules was modulated by the change of pH, and then the endotoxin removal and protein recovery were regulated. The electrostatic interaction and the hydrophobic interaction between the molecules were also regulated by salt concentration. The isoelectric point and the hydrophobicity of the protein had an obvious effect on its recovery. Under the optimal conditions, the recoveries of hemoglobin, human serum albumin and lysozyme were 87.2%,73.4%,97.3%, and the removal efficiencies of endotoxin were 99.8%,97.9%,99.7%, respectively.Finally, the removal of endotoxin from pertussis vaccine was investigated. The effect of medium property (space arm and ligand density), solution conditions (pH value, salt concentration and additives), operation conditions and the different process on endotoxin removal and pertussis vaccine recovery was studied, and then a suitable process was developed. Under the optimal conditions, the endotoxin removal efficiency was above 99%, the protein recovery was 50～70%, the activity recovery of PT was 40～60%, and the activity recovery of FHA was 55～75%.