VIRUS PURIFICATION AND FORMULATION PROCESS
Disclosed herein is provided a virus purification and formulation process for purifying a flavivirus represented by one of a Yellow Fever Virus, Japanese Encephalitis virus, Dengue virus, and West Nile virus. The highly purified flavivirus virus product is characterized as having a low level of sucrose without significant virus loss such as that which is typically encountered by prior art virus purification processes. The disclosed process captures and purifies the virus, separating it from the host cell proteins and DNA, and leaving the host cell proteins and DNA behind. The process also can be used to inactivate and/or concentrate the virus sufficiently for use in formulations.
1 . An essentially pure inactivated flavivirus preparation made by the process of:
a. obtaining a composition suitable for purification from a virus-infected Vero cell culture;
b. clarifying the composition obtained in step “a” by removing cells and cell debris from said composition by depth filtration;
c. subjecting the filtrate in step “b” to endo-nuclease digestion and 0.2 Micron Filtration sufficient to fragment Vero cell DNA followed by filtration;
d. purifying the filtrate obtained in step “c” via a cross-linked agarose matrix with a dextran sulfate functional group chromatography resin and a cross-linked agarose matrix with an octylamine ligand chromatography resin having a shell that excludes molecules with a molecular mass greater than approximately 700 kDa from passing through pores of the shell to achieve depyrogenation of said virus in said filtrate;
e. stabilizing and inactivating the viral product obtained in step “d” with an inactivating amount of ß-PL, and then performing sucrose gradient ultra-centrifugation on the viral product;
f. after inactivating the viral product in step “e”, adjusting the temperature of the viral product to above about 25° C. to about 30° C.;
g. filtering the product obtained in step “f”; and
h. formulating the product obtained in step “g” bound to alum.