title: Safety aspects related to recombinant protein expression from Semliki Forest virus vectors.
abstract: Semliki Forest virus vectors (SFV) have been developed for efficient transgene expression to result in high receptor yields(50-200 pmol receptor/mg protein) in a variety of mammalian host cells. Transfer of the SFV technology to mammalian cells growing in suspension cultures has made it feasible to produce hundreds of milligrams of receptor proteins in a short time. Large-scale production, however, raises the questions of the safety of handling virally infected cells for down-stream processing. Analysis of cell culture medium and SFV-infected cells revealed that some infectious particles were still present. Replacement of virus-containing medium at 2 h post-infection efficiently removed the majority of infectious replication-deficient SFV particles. Washes with PBS further reduced the number of infectious particles significantly both in the medium and associated with cells to levels that allowed safe handling of SFV-infected cells outside the cell culture facility for biochemical, pharmacological, or electrophysiological assays or down-stream processes in connection to receptor purification. Furthermore, engineering of novel temperature-sensitive mutant SFV vectors resulted in temperature-controlled transgene expression, which completely eliminates the risk of contaminating laboratory personnel.
authors.affiliation: Research Laboratories, F. Hoffmann-La Roche, CH-4070, Basel, Switzerland, kenneth.lundstrom@roche.com.
authors.forename: K
authors.initials: K
authors.lastname: Lundstrom
authors.forename: D
authors.initials: D
authors.lastname: Rotmann
authors.forename: D
authors.initials: D
authors.lastname: Hermann
authors.forename: E J
authors.initials: EJ
authors.lastname: Schlaeger