These results are difficult to compare because the standard production methods, standard virus neutralization assays and methods used to quantify the critical viral antigens are not available. Therefore, the production of pure EV71 viral particles as a working standard is critical for EV71 vaccine development. The semi-purified EV71 virion and EV71 virus like-particle can be generated from the harvested virus concentrate by either precipitation with 30% polyethylene glycol or zonal ultracentrifugation using either a 40�C65% discontinuous sucrose gradient or a 5�C40% linear CsCl gradient, but information about the purity and physical structure of these virions is limited. In the present study, we describe the purification and biophysical characterization of EV71 viral particles that were produced from Vero cells grown in a serum-free microcarrier SHP-099 bioreactor system. Several experimental EV71 vaccine candidates are being developed, and encouraging mouse immunogenicity results have been obtained when the prototype vaccine candidates were tested at the preclinical level. Currently, inactivated whole-virion EV71 kb-NB142-70 vaccines appear to be the most potent vaccine candidates; however, immunological results are difficult to compare because the standard production methods, standard virus neutralization assays and the methods used to quantify the critical viral antigens are not available. The whole virion EV71 vaccine was produced by very crude methods, and the downstream purification process and the risk of contamination by host cell proteins were not investigated. Therefore, the biochemical, biophysical and immunological characterizations of purified EV71 viral particles were performed in this study. In addition, we also evaluate whether the purified EV71 viral particles could be used as working standards for EV71 vaccine development. In the current study, a serum-free microcarrier Vero cell culture system for EV71 virus production was established using a low multiplicity of infection to minimize virus seed usage, which could easily be adapted to a large-scale bioreactor process.