title: Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures.
abstract: Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration.
authors: Bayer Biotechnology, Cell Culture and Bioprocess Engineering Dept., 800 Dwight Way/PO Box 1986, Berkeley 94701-1986, California, USA. jens.vogel.b@bayer.com
authors: Jens H
authors: JH
authors: Vogel
authors: Birger
authors: B
authors: Anspach
authors: Karl-Heinz
authors: KH
authors: Kroner
authors: James M
authors: JM
authors: Piret
authors: Charles A
authors: CA
authors: Haynes
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods*; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D006224:Cricetinae; D004867:Equipment Design; D005374:Filtration / Q000295:instrumentation* / Q000379:methods; D008239:Lysine / Q000737:chemistry; D008567:Membranes, Artificial; D011506:Proteins / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D008567:Membranes, Artificial; D011506:Proteins; D010959:Tissue Plasminogen Activator; D008239:Lysine