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 (CEF) 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 purification of cell culture derived VV after an initial host cell homogenization and clearance centrifugation. This affinity chromatography is based on the interaction between the VV surface protein A27L and heparin, which is currently further characterized by surface plasmon resonance technology. In addition to heparin are heparin like molecules investigated. Small scale chromatography experiments with a heparinized polymer resin (Toyopearl AF-Heparin) and heparinized reinforced cellulose membrane have proven to be a suitable matrix to capture VV. However, the heparinized cellulose membrane has a far higher binding capacity than the heparin polymer resin. This is mainly due to the size of the VV virions, which are brick shaped with a diameter of 250 to 350 nm. Therefore, the virions cannot penetrate the pores of the bead based resin. Hence, membrane affinity chromatography represents a valuable choice to capture virus particles in addition to the general advantages of membrane chromatography, like lower pressure drop, ease of scale up, lower processing time and higher concentration factor. Moreover, we have compared classical ion exchange membrane chromatography and cellufine sulfate with heparin derivatized membrane chromatography and found a far higher removal rate of contaminating host cell proteins and DNA with the heparinized reinforced cellulose membrane.