(726e) Capturing of Cell Culture Derived Vaccinia Virus by Membrane Adsorbers

Smallpox is an acute, highly infectious viral disease unique to humans with a mortality rate around 25%. It is caused by the Variola virus that belongs to the family of Poxviruses. Smallpox was responsible for an estimated 300-500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, the World Health Organization (WHO) certified the eradication of smallpox in 1980. After the eradication, the compulsory vaccination was abandoned – with the result that about half of the world's population is not vaccinated. This represents a potential threat in the case of a deliberate release of Variola virus as an act of bioterrorism. Consequently, several governments are ordering stock piles of smallpox vaccines to protect their populations from this remote, but extremely grave threat. MVA-BN® is a third generation smallpox vaccine based on the Modified Vaccinia Ankara (MVA) virus which demonstrates superior safety compared to traditional smallpox vaccines based on native Vaccinia virus (VV) strains. In addition, re-engineered VV represent as a robust vector a platform technology for vaccine delivery systems as e.g. in the case of HIV, Dengue fever, Japanese encephalitis and cancer.

Traditionally, VV- and MVA virus based vaccines have been grown in primary chicken embryo fibroblast cultures and purified either by sucrose cushion or sucrose gradient centrifugation, or by ultrafiltration. However, a potential shift from primary to continuous cell cultures would impose stricter requirements regarding the purity level of the vaccines, and a new generation of vaccine manufacturing processes is needed that include more sophisticated and innovative downstream techniques for purification.

Here, we report the development of an affinity chromatography of cell culture derived VV after an initial host cell homogenization and clearance centrifugation. The Vaccinia viral envelope protein A27L is known to bind to heparin.. Based on this, small scale chromatography experiments with heparinized polymer beads and cellufine sulfate beads in addition to heparinized cellulose membranes have been conducted. There we have found that membrane adsorbers are superior over bead based chromatography media in terms of efficiency and productivity.

Subsequent studies compared ion exchange membrane adsorbers with a heparinized membrane adsorber. The results indicate that the overall performance of the affinity chromatography in terms of virus capturing and contaminant removal is better than any of the tested ion exchange membrane adsorbers.

Hence, membrane affinity chromatography represents a valuable choice to capture VV particles in addition to the general advantages of membrane chromatography.