The Purification Of Inactivated Foot-and-mouth Disease Virus Antigen By Ion-exchange Chromatography

Inactivated foot-and-mouth disease virus(FMDV)vaccine is one of the most important vaccines for preventing foot-and-mouth disease,which is a highly infectious disease of ruminants and would result in large economic loss once breakout.Purification is a key step to ensure sufficient immunogenic effect and safety for inactivated FMDV vaccine.Chromatographic techniques possess advantages of scalability and high selectivity,thus providing an alternative strategy for FMDV vaccine purification.It is challenging to obtain high recovery and stable native structure for inactivated FMDV whose size is about 28 nm during chromatography.In this paper,we have investigated the critical factor on purification efficiency and structural stability for purification of inactivated FMDV by ion exchange chromatography(IEC),and tried to establish a novel purification process for industrial production,including:(1)Three anion-exchange media with similar particle size and ligand density except pore size,including DEAE-FF(32 nm),DEAE-650M(106 nm)and DEAE-POROS(214 nm),were applied and compared for purification of inactivated FMDV.We have investigated the effect of pore size of media on the dynamic binding capacity(DBC)for inactivated FMDV,and results show that the DBC for DEAE-POROS and DEAE-650M were 11.53 and 10.03 mg/mL,while that for DEAE-FF was less than 1/10 of the previous two.And confocal laser scanning microscopy(CLSM)also confirmed that inactivated FMDV mostly confined to a thin shell on the outer surface of DEAE-FF,but permeated into intraparticle region of DEAE-POROS.(2)The effects of pore size of media on the recovery and stability of inactivated FMDV were investigated during IEC process.The recovery of inactivated FMDV after chromatographic process was 68.42%,66.32%and 54.46%on DEAE-POROS,DEAE-650M and DEAE-FF in sequence.High performance size exclusion chromatography(HPSEC)analysis results indicated that interaction between inactivated FMDV and media was the main reason for FMDV dissociation into smaller subunits 12S,and severer dissociation occured on media with smaller pore size.Possible denaturation of the FMDV on the surfaces of anion exchange media was analyzed by differential scanning calorimetry(DSC).The transition temperature(Tm1),dissociating of inactivated FMDV into 12S,is at about 48.52? in solution.When inactivated FMDV were absorbed on DEAE-FF,the Tm1 became 41.73?,indicating increased the possibility of dissociation.The Tm1 for DEAE-650M and DEAE-POROS was 44.04? and 45.37?,showing less dissociation and higher thermostability was obtained on DEAE-POROS.(3)We developed a purification process for inactivated FMDV by DEAE-POROS.Inactivated FMDV crude was loaded onto DEAE-POROS IEC at the optimum condition,including conductivity of buffer,the protein concentration of crude and retention time.94%inactivated FMDV recovery was reliably recovered from crude solution by IEC.After polished by ultrafiltration and size exclusion chromatography(SEC),intact inactivated FMDV with high-purity was obtained.The average total recovery was 79%,with 173-fold increase in purity,and above 95%host DNA was removed.In summary,this work demonstrated that media with large pore size was superior to the conventional agarose-gel based chromatographic media for the purification of inactivated FMDV in several aspects,including static and dynamic binding capacity,thermostability and recovery during chromatographic process.And the mechanism of dissociation of inactivated FMDV on DEAE media has also been investigated.All these results will provide a useful guidance to establish an efficient purification process for FMDV.