Study On Methods And Requirments For The Quality Standard Of PEGylated Trichosanthin Injection

In order to develop the quality control methods and the quality standard of PEG-TCS and its injection.First of all,according to the determine methods of TCS,the requirement of China pharmacopoeia along with PEG-TCS character and the modifying course,the quality control methods were studied and the standards were studied out in the following aspects: HPLC methods has been setted up and validated;PEG dye method has been setted up to determine the free PEG remain;CBBG-250 dye was used to determine the NaCNBH₃ remain,and it was compared to the iodine titration method;The Lowry method has been validated for the protein content of PEG-TCS;The abortifacient activity of PEG-TCS was analysed on above results and China pharmacopoeia,PEG-TCS quality control standards were setted up and three batch samples were determined following the methods:The protein has been identified ace.Basing ording to the metods of Western bloting and PEG dye,The PEG-TCS molecular weight was determined by deoxidized SDS-PAGE,whether NaCNBH₃ remain,the residual free PEG content,were determined by CBBG-250 dye and PEG dye method respectively,the protein content was determined by Lowry,and free TCS was determined by the HPLC and SDS-PAGE.The abortifacient activity of PEG-TCS was analysed.Besides the injection of PEG-TCS was also analysed in endotoxin content,pH,the isoelectric point,asepsis and so on according to the correlation requirements.A TSK-G3000SW column(7.5×300mm)was used.The mobile phase was 0.05mol/L NaH₂PO₄-0.3mol/LNaC1-95%Alcohol,the detection wavelength was 222nm,the column temperature was 25℃.The calibration curve linear within the range of 5μg/ml～50μg/ml for TCS(r=0.9992,n=5).The average recovery is 99.96%(RSD=1.8%).TneTCSremai by HPLC is≤5μg/ml,residual free PEG contentis≤15μg/ml.In the PEG-TCS injection,no NaCNBH₃ remains,the protein content is about 1.0mg/ml±0.15 mg/ml.Biological activities in vivo was enhanced.The metods of Westem bloting and PEG dye were both masculine.PEG-TCS molecular weight is 54KD±5.4 KD.The PEG-TCS is transparenct liquid,pH is 3.0-5.5.The asepsis experiment,the endotoxin respond experiment both conform to the requirements.In conclusion,the experiment data for forming standard of PEG-TCS was provided, at the same time the quality standard can be used to evaluate and control the quality of PEG-TCS effectively.PEG-TCS and its injection in content,relate substances,endotoxin content,asepsis and so on conform to the quality requirements under the new drug.