Purification Technology Research On Inactivated Japanese Encephalitis Vaccines Produced By Vero Cells

ackground: Previous clinical studys of Japanese encephalitis virus (JEV) vaccineshowed that human bodies have a good tolerance towards antigens of inactivated JEV vaccineand showed mild adverse reactions, and a handful of serious adverse reactions wereassociated with other components in inactivated JEV vaccine rather than antigens. Thesecomponents including residual protein and DNA of cells which used for viral propagation,residual components in culture medium such as calf serum, residual inactivators (likeformaldehyde), additive excipient such as gelatin, antiseptic such as merthiolate and et al,[15-20]Therefore, improving the purity of antigen and avoiding addition of inappropriateexcipient, protective agent and preservative, may likely improve the safety of vaccine.Objective: The existing purification processes of JEV vaccine are generally the zonecentrifugal technology and multi-step gel filtration technology. The biggest problem of thosemethods is the existence of excessive amount of hazardous substance which leads to potentialrisk of safety of vaccine products. Hence, a systematic research on antigen purificationprocesses of JEV vaccine was conducted in this study to improve the purity of vaccine andthen reduce adverse events following immunization.Methods:(1) Study on Inactivation processes by comparing the inactive effects ofmethanol and β-propiolactone on theJEV;(2)Study on concentration processes by comparingthe influence of ultrafiltration membrane bags with different aperture and different cycles ofconcentrations on concentration;(3)Study on purification by using two molecular sieve filler,Sepharose4FF and Sephacryl S-300;(4)Evaluating the effect of ion exchangechromatography on the purification of Japanese Encephalitis virus;(5) Optimization approachis used to purify three batches of laboratory-scale samples and three batches of pilot-scalesamples.Results:(1)Compared with methanol, β-propiolactone has an better recovery rate andlow cost of production as an inactivator of JEV;(2) Using membrane of300K apertureconcentrated for20times can get a relatively high antigen recovery rates and decreaseprocessing volume of purification, and it was used in following stage;(3)Sepharose4FF chromatography can achieve a relatively higher purified sample, while Sephacryl S-300chromatography sample has a higher antigen recovery rate. Although both of them fail tomeet the requirement of one single step purification in pharmacopeia, we prefer to useSephacryl S-300with a higher recovery rate as first step of essence purification;(4) Theresidue of host DNA is less than10pg/dose, both the residue of Vero cellular protein and BSAare over10times less than2010pharmacopoeia standard when ion exchange chromatographyis used;(5)Purified original liquid extracted from three batches of small-sized samples andthree batches of pilot-scale samples meets standard of stability.Conclusion: When ion exchange chromatography added, the purity of antigen isincreased dramatically. Meanwhile, it makes the residue DNA below10pg/dose, as well asresidue amounts of both the Vero cell protein and the bovine serum albumin are10times lessthan the amounts required by Pharmacopoeia of2010edition and also are significantly lowerthan those products in the domestic. That improves safety of the vaccine and also keeps theexcellent immunogenicity. Products with high quality will definitely lower probability ofoccurrence of side effects and be popular among parents and medical staffs, since children aremajor inoculator of Japanese Encephalitis vaccine. In the meantime, it can create relativelyhigh social and economical benefits.