Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography.
A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%.
Wolff
Michael W
MW
Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse. 1, Magdeburg, Germany. mwolff@mpi-magdeburg.mpg.de
Siewert
Corina
C
Hansen
Sara Post
SP
Faber
Rene
R
Reichl
Udo
U
D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008566:Membranes; D014616:Vaccinia virus / Q000254:growth & development / Q000302:isolation & purification*