Establishment and Verification of a Method for Detecting Trypsin Residues

Abstract

Establish a detection method for residual trypsin and validate the method. Using enzyme-linked immunosorbent assay (ELISA) to detect residual trypsin in the single virus harvest solution and stock solution of oral trivalent reconstituted rotavirus attenuated live vaccine (Vero cells) (hereinafter referred to as trivalent rotavirus vaccine). At the same time, according to the requirements of Part 9101 of the Chinese Pharmacopoeia (2020 edition), the specificity, accuracy, repeatability, intermediate precision, linearity, durability, quantification limit and other indicators of the method are evaluated. The specificity verification results show that the recovery rates of recombinant trypsin standards at each concentration level are within the range of 70% to 125%, and the impact of MEM on the recovery rates of standards can be ignored. The accuracy verification results show that the recovery rates of the three concentrations of high, medium, and low (5ng/mL, 2.5ng/mL, 1.25ng/mL) diluted standard samples are all between 70% and 125%. The repeatability verification results showed that the relative standard deviation (RSD) of the six measurements of the trivalent rotavirus vaccine stock solution and the single virus harvest solution were both less than 15%. The intermediate precision validation results showed that the relative standard deviation (RSD) of the six measurements conducted by the two researchers on the trivalent rotavirus vaccine stock solution and the single virus harvest solution were both less than 15%. The standard curve plotted by the Logistic five parameter fitting curve calculation method based on the results of three freeze-drying standard sample tests for linear validation has a correlation coefficient R2 greater than 0.98. The durability validation results showed that there was no significant difference (P>0.05) between the three enzyme-linked immunosorbent assay (ELISA) results under 36℃ and 38℃ conditions and those under 37℃ conditions. The quantitative limit validation results show that after diluting the freeze-dried standard to the theoretical minimum value, the bottom limit value that is closest to the theoretical value and has the highest recovery rate is 0.312ng/mL. The enzyme-linked immunosorbent assay (ELISA) is suitable for the determination of residual trypsin in the single virus harvest solution and stock solution of trivalent rotavirus vaccines.