Establishment of a method for potency determination of reteplase by automatic coagulation analyzer

WANG Lü-yin; BAI Bi-wei; XU Kai-xin; WANG Yu-chen; LÜ Ping; LI Yi; LI Jing

doi:10.20251/j.cnki.1003-3734.2025.20.011

Abstract：Objective:To establish a potency determination method of reteplase(rPA)by automatic coagulation analyzers.Methods:The coagulation method of automatic coagulation analyzer was adopted.Sample or standard series dilution,thrombin(intermediate reagent),fibrinogen and plasminogen mixture(initial reagent)were added in turn.The reaction temperature was 37℃,the primary wavelength was 405 nm,the standard time was 900 s,and the delay time was 10 s.Threshold(percentage of curve)was set as the primary algorithm.The lysis time of rPA standard solution with five concentrations(600,800,1 000,1 200,and 1 400 ng·mL-1)and sample solution with three concentrations(800,1 000,and 1 200 ng·mL-1)was determined.The concentration and potency(U·mL-1)of the rPA sample were calculated by standard curve.Specificity,accuracy,linearity and range,precision and durability were systematically investigated in a laboratory.The potency of two batches of rPA for injection and one batch of rPA stock solution were determined by automatic coagulation analyzer method and bubble-rising method,respectively.Results:The rPA standard curve was Y=-0.5887X+4.508,R2=0.9957.Neither human serum albumin nor the formulation solution of rPA showed fibrinolytic activity,and the specificity of the method was good.The intermediate precision(CV,%)and repeatability(CV,%)of the five potency levels(60%,80%,100%,120%,and 140%)were all less than 2%and the relative bias of each potency level was in the range of 0.29%～1.01%,demonstrating the good accuracy and precision of the method.The automatic coagulation analyzer method can effectively detect the potency of rPA stock solution and preparation.There was no significant difference between the automatic coagulation analyzer method and the traditional bubble rising method.Furthermore,automatic coagulation analyzer method performed better precision.Conclusion:The automatic coagulation analyzer method was established for the determination of potency of rPA,which has the advantages of simple operation,short time consumption,high accuracy and precision.Compared with bubble-rising method,it has obvious advantages and can be used as efficacy evaluation in-vitro and quality control of rPA products.