Establishment and evaluation of analytical method of antigen purity in the EV71-CA16 bivalent inactivated vaccine of HFMD by HPLC-UV
To establish a relatively accurate and reliable HPLC-UV method for the purity detection of relevant antigens in EV71-CA16 bivalent inactivated vaccine of hand-foot-mouth disease(HFMD):Entero virus 71(EV71)and Coxsackievirus group a type 16(CA16).HPLC analysis was performed on a Waters volume exclusion chromatography column(SEC,Ultrahydro-gelTM 250 7.8×300 mm,6 μm).The mobile phase consisted of 0.01 mol/L PBS and 0.1 mol/L NaCl with equivalent elution.The flow rate was 0.3 mL/min,the detection wavelength was 280 nm,the injection volume was 50 μL and the column tempera-ture was 21℃,were used to detect antigen purity of EV71 and CA16 virus drug substances,and the detection limit,tailing factor,specificity,repeatability and detection range of the method were verified.The results showed that the signal-to-noise ratios(S/N)of the sensitivity solutions for the two antigens were 37.053 and 47.710,respectively,those were both greater than 3,and the tailing factor of each specimen was less than 3.The peak fractions of the CA16 stock and EV71 stock tar-get antigen after purification by HPLC were specifically examined by SDS-PAGE and Western-blot,those results showed that the chromatographic peak sample composition were identified as the structural proteins of the capsids of the two viruses,CA16 and EV71,respectively,indicating a reliable specificity.After multiple testing with different concentrations of EV71 and CA16 viral antigens,the coefficient of variation RSD between the peak area percentage and retention time at each con-centration condition was less than 1％,indicating acceptable repeatability.The test results of samples of different antigen con-centration batches showed that,the antigen concentration of CA16 stock solution was between 89 U/mLand 26 398 U/mL and the antigen concentration of EV71 stock solution was between 170 U/mLand 9 074 U/mL,both antigen solutions statistically showed less than 1％RSD of peak area and retention time after multiple testing,indicating this assay was accurate and reli-able at such antigen concentration,the HPLC-UV method established in this study could be used to detect the virus antigen purity in EV71-CA16 bivalent inactivated HFMD vaccine..
HPLC-UVEV71-CA16 bivalent inactivated vaccine of HFMDPurity determinationEnterovirus 71(EV71)Coxsac-kievirus A 16(CA16)
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