Development And Application Of New Technologies For Quality Control Of Inactivated Foot And Mouth Disease Virus Vaccine

Efficient quality control(QC)during vaccine processing plays a critical role for production of high-quality vaccines and for prevention of infectious diseases.For many years,the QC has been an important yet difficult subject because of the problems in characterization of the viral antigens that are tens to hundred nm in size,very complex in three-dimensional structure and very low in solution concentration.Their biological activities are strongly dependent on their structural integrities.Traditional vaccine QC methods,such as in vivo animal test and in vitro immunoassays,are costly,time-consuming,and inaccurate.In this work,new QC techniques for inactivated foot and mouth disease virus(iFMDV)vaccine,were designed and developed,including nuclease digestion-high performance size exclusion chromatography(ND-HPSEC),capillary zone electrophoresis(CZE)identification,and differential scanning fluorimetry(DSF)measurement.These technologies enable both the quality control and the in-situ investigation for product improvement and stabilization of viral vaccinesThe results and novelties are as follows(1)A ND-HPSEC was developed for qualification of 146S in commercial iFMDV vaccines.Four pre-analytical treatment methods,including ultracentrifugation,PEG precipitation,centrifugal chromatography and nuclease digestion(ND),were investigated systematically and compared for their removal efficiency of the interfering impurities in iFMDV vaccines.Among all of them,ND was the best for its advantages including efficient removal of the interfering impurities,fast processing speed,and mild operating conditions.The optimal condition for ND using Benzonase determined by response surface method was as follows:appending Benzonase into 200?L of antigen phase to a final concentration of 421 U/mL and incubating at 25.1? for 1.29 h.The developed ND-HPSEC was then applied for rapid analysis of 12 bathes of commercial iFMDV vaccines products with different serotypes produced by 4 different manufacturers.Results showed the method was applicable to various iFMDV vaccines with good reproducibility(RSD<5.3%,n=3).The pre-analytical treatment method based on ND removed interference from impurities during quantification of 146S,and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability(2)A simple and rapid capillary zone electrophoresis(CZE)method was developed for on-line separation and quantification of iFMDV antigens in monovalent and bivalent iFMDV vaccines.After optimizing CZE conditions including UV detecting wavelength,background electrolyte,injection volume,and separation voltage,both serotype A and O 146S showed good reproducibility(RSD<5%)and linear responses(R2=0.999)between the peak area and 146S content in the concentration range of 15-400 ?g/mL.The two serotypes of 146S with similar size had different migration times in CZE due to difference in their zeta potentials,which allows them to be separated and quantified,with accuracy of<10%relative error.CZE was then successfully applied for antigen quantification of commercial O monovalent and A/O bivalent iFMDV vaccines.Compared with HPSEC,CZE was not only able to quantify each serotype of 146S,but also free from interference of nucleic acids impurities(3)DSF was employed for on-line analysis of thermal stability of 146S in three representative adjuvants including aluminum hydroxide(AH),oil-in-water(O/W)emulsion,and water-in-oil(W/O)emulsion.Using SYBR Green ? as the fluorescent probe,DSF enables detection the Tm of iFMDV at concentration as low as 5 ?g/mL The Tm of iFMDV in all three adjuvants were slightly lower than that in buffer solution,among which the Tm of iFMDV adsorbed on aluminum hydroxide was the lowest Screening for excipients in different adjuvants was successfully conducted using DSF Sugars and glycerol increased the Tm of iFMDV in all three adjuvants,but to different degree.The screening results were further verified by differential scanning calorimetry(DSC)and HPSEC.Moreover,DSF was proved also applicative for low-purity iFMDV and pre-adjuvanted iFMDV vaccines,without being interfered by other protein impurities.(4)The stabilizers of 146S in ISA 206,a complex water-in-oil-in-water emulsion adjuvant,were screened by HPSEC and DSF.After screening for buffers,excipients and metal salts,it was finally determined the stabilizers were 200 mM HEPES solution containing 65 mM CaCl2 and 50 g/L sucrose for both serotype A and O 146S.In this stable condition,Tm of serotype A and O 146S was 59.2? and 58.2? respectively,which was nearly 10? and 12? higher than that of the control group.After emulsified with ISA 206 and stored at 37?,the residual antigens of serotype A with stabilizer was about 35%after 50 days,while that of the control was less than 10%.For serotype O antigens,which was more unstable,almost no dissociation occurred with stabilizer after 40 h,while only 7%of antigens were remained in control(5)Combined with animal tests,the effect of size exclusion chromatography(SEC)process on the structure of iFMDV was investigated by many quality control techniques,including HPSEC,DSF,CD,and fluorescent probe diffusion.Strategies to inhibit the structural changes of iFMDV were then explored.It was found that after the SEC processes with media pore size close to or larger than particle diameter of iFMDV,there was no apparent change in the size and molecular weight of iFMDV.However,their retention time in HPSEC chromatograms advanced noticeably,accompanied with slight changes in secondary and tertiary structures,as well as a decrease in Tm value Fluorescent probe diffusion experiments suggested the virus particle became less rigid and its structure was more incompact.In contrast,none of these change was detected for iFMDV which was after the SEC processes with media pore size smaller than their particle diameter.Based on this findings,it was speculated that in cases that iFMDV could diffuse into the medium pores during SEC,there were physical collision between iFMDV and the inner wall of the medium,thus led to slight structural change in iFMDV Animal tests indicated that such structural change and decrease in stability also caused decrease in induction of iFMDV specific antibodies in immunized mice,especially in the early stage of immunization,which is not conducive to the rapid formation of immune protection after vaccination.Adding 5 mM CaCl2 to SEC mobile phase was found to inhibit the destruction of 146S structure effectively.