Establishment And Evaluation Of A Quantitative Method For Bacterial Endotoxin

BackgroundBacterial endotoxin is the main heat substance in injections,which can cause fever,disorder of microcirculation,endotoxic shock and DIG.The reliability of the test's result is directly related to the sensitivity and reliability of endotoxin detection method.At present,we use gel method,which is only for the limit judgment.Reliability and accuracy is the basic standard in vaccine quality control,so it is necessary to establish a quantitative method of bacterial endotoxin.MethodBased on the Principle 1143 "Bacterial Endotoxin Test"(?China Pharmacopoeia?2015 edition),this study will analyze the results of Bacterial endotoxin from gel method.According to this analysis,a Kinetic turbidimetric method will be established,to provide a quantitative analytical method.The methodological validation includes accuracy,precision,specificity,limits of quantitation,linearity,range,and durability.After the method validation is completed,each sample will be tested,to confirm the adaptability of the samples.According to the results of adaptability tests,possible interference and interference cancellation in each sample will be tested and analyzed,And determine the dilution multiple of each sample make a primary evaluation for its actual applicational.ResultKinetic turbidimetric method was established and the results are as follows:(1)Accuracy:the average recovery rate of each group is 100±30%,and it meets the required standards of 50%-200%.(2)Precision:Three different concentration are prepared,and each concentration should be measured 3 times,the RSD of the same concentration are no larger than 1.The same sample are used for six more parallel tests.This testing results indicate that RSD is no larger than 0.85,that means it meets the requirement,that the RSD should not be greater than 1.(3)Specificity:Five important samples will be tested,including the fluid of IPV and EV71.IPV.EV71 and fHAV.The results indicate that this method can't be affected by impurities and can accurately detect the bacterial endotoxin of them.(4)Quantitative limit:According to the range of sensitivity,three low concentration(0.015EU/ml?0.020EU/ml and 0.025EU/ml)are selected for the experiment.The recovery rate was 75?110%,and it meets the standard of 50%-200%,the quantitative limit is up to 0.015EU/ml.(5)Linear(standard curve reliability):4 experiments are performed,each with different lowest concentration and different multiples,R2:0.994,0.986,0.982 and 0.988,it met the standard of R2>0.98.(6)Range:Within the sensitivity range of 0.015EU/ml?10.0EU/ml,the recovery rate is between 90%to 120%,fulfills the criteria.According to the results of test±20%sensitivity range,the upper limitation of detection can be extended appropriately to 12.0EU/ml,but the lower limit cannot be extended anymore.(7)Durability:The contents of different proteins are prepared,as are the different pH and different production processes.pH of EV71 are 6.8,7.2 are artificially regulated to 8.5;different proteins of sIPV of 2.3ug,3.3ug and 3.9ug.purified and fluid from this batch are also tested.The results indicate no differences between the data.The results are unaffected and meet the requirements of durability.The systematic Testing of the samples of endotoxin needed vaccines indicate following results:the recovery rates and adaptability tests of each sample fulfill the criteria,which means that this certain method is adaptable to the manufacture by the institute of medical biology chinese academy of medical science and the results are accurate and reliable.By increasing the sample dilution,this study solved the problem,that animal samples like bovine serum tends to be easily prone to positive interference.ConclusionThis study established a Kinetic turbidimetric quantitative method of bacterial endotoxin successfully.The methodological validation meets the criteria of the Chinese Pharmacopoeia"guidelines for the analysis and validation of drug quality standard”.The Testing results fulfill the standards.Each vaccine products are tested by this method,and the adaptability of the various samples are benign.So the method can be used for routine testing.