Study on purification of hepatitis A virus strain HM-175 from primary monkey kidney cell culture of Maccaca mulatta for preparation of an inactivated hepatitis A vaccine

ABSTRACT

Monolayer primary monkey kidney cell culture was infected with vaccine production hepatitis A virus strain HM-175 at a multiplicity of infection (MOI) of 0.05 PFU cell and incubation period was 21 days. Then the culture was stored at 70oC. Freeze and thaw 3 times for cell breakage and virus release. Centrifugation for separation of cell debris, then cell debris was sonicated with 3 cycles of 30 second and 30 seconds intervals, centrifugation separation of cell debris. Cell culture supernatant and supernatant after centrifugation were mixed for further ultrafitration and concentration using filtration system pellicon following one rate-zonal ultracetrifugation in sucrose gradient solution were conducted for HAV separation and purification. A purified solution of HAV antigen including empty and full particles was obtained by using this method, which was further, treated with 1/2000 formalin for HAV inactivation at 35oC - 96h. Hepatitis A vaccine prepared by this method meets the WHO minimum requirement for this vaccine.