The present invention provides novel serum-free cell culture medium and
methods for cultivating MDCK cells. In particular, non-tumorigenic MDCK
cells. The present invention also provides methods for producing
influenza viruses (e.g., particularly cold-adapted, and/or temperature
sensitive, and/or attenuated influenza viruses) that eliminate the need
for a cell culture medium exchange step. The novel medium and methods are
useful to grow influenza viruses, in cell culture to high titer. The
present invention further provides purification methods for purifying
influenza viruses with high overall recovery of live virus and result in
levels of host cell DNA (HCD), host cell protein (HCP) and non-specific
endonuclease (e.g., Benzonase), which are below the specifications
required by regulatory agencies. The immunogenic compositions can be used
to actively immunize subjects or to generate antibodies for a variety of
uses, including passive immunization and diagnostic immunoassays.