Synth Study On The Purification Process Of The Coxsackievirus A16(Vero Cell) Vaccine

Base on the date of Chinese CDC,the cases of HFMD were consistently more than half of the class C infectious diseases since the monitor of HFMD.The vaccine that can control the HFMD is urgently needed.However,the CVA16 vaccine has not yet been applied to clinical trials.This study was on the initial stage of the development of CVA16 inactivated virus vaccine,aiming to obtain high purity virus.We hoped to explore the route and suitable parameters of CVA16 vaccine for mass purification and the subsequent study of CVA16 vaccine purification process.The CVA16 virus bulk and a large number of virus particles were purified.The gel chromatography and ion exchange were used in the purification steps.The gel chromatography was used to study the selectivity,separation effects and high recovery rate of virus particles.The sampling time and sampling area were determined basing on the characteristics of the separated substances in the sample.An anion exchange filler was selected.The separation effects of the filler were studied in the experiment.The sample volume and the flow velocity were selected.The samples collected at the optimum condition were analyzed using methods of HPLC,western-blot,SDS-PAGE and electron microscope.The results were as follows : 1)the conditions were obtained in the preliminary purification processes :1)The CVA16 antigen was found in the first flow-through protein peak,and the collection time was from 19 minutes to 30 minutes,and theretention time was 11 minutes.Six batches of CVA16 vaccine bulk were producted,the antigen recovery rate of the virus through gel chromatography reached 40% to 50%,the recovery rate was basically stable.2)the conditions were obtained in the fine purification processes : the flow rate is 1ml/min by using anion gel chromatography and the sampling amount is 20%CV.The critical process parameters and acceptance criteria were as follows:(1)The limit of bovine serum albumin was below 50 ng/ml;(2)Peak area of particle sample accounted above 95% total area and the higher antigen recovery rate was obtained using HPLC method.CVA16 antigen was presented in the first flow protein peak,so the sample time was mainly from 19 minutes to 30 minutes,and the retention time was 11 minutes.Referred to the similar verificated vaccine requirements in Chinese pharmacopoeia(2015 edition)and the CFDA reference criteria,this standard was applicable to the evaluation of vaccine quality.