Research On The Purification Of Poliovirus Sabin Strains By Two Kinds Of Gel Filtration Media

Poliomyelitis is widely identified to be an acute viral infectious disease with highly infectious and extremely harmful caused by poliomyelitis virus(PV).There is no effective treatment for the disease and the only way to prevent people from being infected is vaccination.Oral poliomyelitis vaccine(OPV)was widely used due to its convenient and cheap,but the vaccine-associated paralytic poliomyelitis(VAPP)and vaccine-derived poliovirus(VDPV)cases caused by OPV use have increasingly received great attention.Inactivated poliomyelitis vaccine(IPV)can avoid VAPP and VDPV,to gradually replace OPV with IPV for avoiding the VAPP and VDPV,becomes the emphasized issue in the Polio Eradication and Endgame Strategic Plan.After the eradication of polio around the world,the conventional IPV production is generally carried out facilities with bio-containment requirement more stringent.WHO encourages developing the IPV made from Sabin strain(sIPV).According to our current production process,we purified the Sabin poliovirases concentrated using Sepharose CL-6B after the harvesting,clarification and concentration of viruses.It could be a time consumed-and plant capacity limited course.Thus,a more effective manufactural method is urgent needed.In this study,we.used two kinds of gel Sepharose CL-6B and Sepharose 6FF to purified Sabin polioviruses in a 3%or 6%column volume respectively.We comparatively analysis the purification results by detection of gel filtration chromatography and ion exchange chromatography of relevant indicators.After that,we validated the effect of two kinds of gels used for polioviruses purification.Our data could be a support to sIPV production process improvement.Objective:To optimize the production process,we used two different kinds of gel chromatography media to purify Sabin poliovirus,after that we tell the better purification scheme.Methods:Chromatography media Sepharose CL-6B and Sepharose 6FF were respectively applied to purify type ?,? and ? Sabin polioviruses which are concentrated. Firstly,we collected all the outcomes in all of the purification phase.Then we tested D antigen and protein content of all the collections in order to confirming the collection of viruses.To those collections contained the highest D antigen,we purified them with ion exchange chromatography DEAE Sepharose FF.Further,D antigen content in viruses harvesting fluid,Concentrates or chromatography collections were evaluated by ELISA,through which the recovery rate of D antigen were calculated.According to the Chinese Pharmacopoeia,we measured the total protein content in viruses harvesting fluid,in the liquid of gel filtration purification and got the specific activity and the Protein removal rate.Finally,it had been done in a recommended way by Chinese Pharmacopoeia to detect the Vero cell DNA residue,Vero cell protein residue,bovine serum albumin residue and antibiotics residue in the purified viruses.Results:When we purified the samples in 6%Sepharose 6FF gel column volume,the average recovery rate of 3 types of viruses harvesting fluid filtered was 78.69%,78.03%,78.37%,respectively.The recovery rate of Sepharose 6FF was the same as the antigen average recovery rate of Sepharose CL-6B which was purified the samples in 3%for 77.29%;78.60%,77.36%(P>0.05).There is no significant difference of the specific activity of each type viruses harvesting fluid purified between Sepharose 6FF and Sepharose CL-6B.Other kinds of tests of flitered harvesting fluid and viruses purification fluid purified by these two gels accord to the national standard and no major difference.Conclusion:There was no significant different between antigen recovery rate and protein specific activity of each type of antigen purified fluid purified by Sepharose 6FF and purified by Sepharose CL-6B.However,using Sepharose 6FF could be a time saving way.Thus,it is better to replace Sepharose CL-6B with Sepharose 6FF.