Lyophilization Of Hemoglobin-based Oxygen Carrier And Storage Of The Freeze-Dried Solids

Hemoglobin-based Oxygen Carrier(HBOCs) can carry and unload oxygen,it has been widely tested in hemorrhage shock,enhacing the chemotherapy effect,improving microcirculation and so on.However,such solution is unstable,there is marked rise in metHBOCs content upon storage.To facilitate its storage,HBOCs solutions were freeze-dried with addition of various kinds of protective agents to control the formation of metHBOCs.The contents of the thesis fall into 2 parts:firstly,in order to screen the good protective agents for freeze-drying, HBOCs were lyophilized together with different kinds of protective agents;secondly, freeze-dried solids were treated under different conditions of temperature(4℃,room,40℃, 60℃) and time of storage(up to 4 months ) to test their stability.Meanwhile,the characteristics of the lyophilization products were determined.Result:Stabilization of 300kD or 64 kD HBOCs were achieved by 0.22M glucose, 0.11M trehalose,0.11M sucrose,or 0.2M amino-acid salts during freeze-drying.The UV spectrum,SDS-PAGE and SEC of the dried solids were recorded and they were nearly the same as that in original solution.In addition,relations between concentrations of monosaccharide/disaccharide and the methemoglobin level before and after lyophilization were calculated.When the concentration of protective agents is≥3%,the methemoglobin level did not increase as compared with that of control group(40g/L Hemoglobin-based oxygen carriers before freeze-dried).Long time storage experiment:according to metHBOCs content for clinical experiment standard,dried solids can storage at room temperatureat at least for one month or at 4℃for at least 4 months.During storage,the UV spectrum,SDS-PAGE and SEC of the dried solids were nearly the same as that in dried solids before 4 months.Destructive tests of freeze-dried products of 64kD at 60℃and freeze-dried solids of 300kD at 40℃showed that 2%glucose and 2%dextran-trehalose complex were the optimum protective agents for the lyophilization of the HBOCs.